Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THE INVENTION
INHIBITORS OF FARNESYL-PROTEIN TRANSFERASE
BA.CKGROUND OF THE INVENT~ON
The Ras proteins (Ha-Ras, Ki4a-Ras, Ki4b-Ras and N-Ras~
are part of a si~n~llin~ 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 inclllced to exchange GDP for
GTP and undergoes a conformational change. The GTP-bound form of
Ras propagates the growth stim~ tory signal until the signal is
termin~ted by the intrin~ic 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-tr~n.~l~tional
modifications are involved with Ras membrane loc~li7~tion, and all 3
modifications occur at the C-termin~l~ of Ras. The Ras C-telll~illus
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, which catalyze the aLkylation of the cysteine residue of the
CAAX motif with a Cls or C2~ isoprenoid, respectively. (S. Clarke.,
Ann. Rev. Biochem. 61:355-386 (1992); W.R. Schafer and J. Rine, Ann.
Rev. Gene~ics 30:209-237 (1992)). The Ras protein is one of several
proteins that are known to undergo post-translational farnesylation.
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Other farnesylated proteins include the Ras-related GTP-binding
proteins such as Rho, fungal mating factors, the nuclear l?~min~, 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
S farnesylated 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-transforrned cells in soft agar and to modify
other aspects of their transformed phenotype. It has also been
demonstrated that certain inhibitors of farnesyl-protein transferase
selectively block the processing of the Ras oncoprotein intracellularly
(N.E. Kohl et al., Science, 260:1934-1937 (1993) and G.L. James et al.,
Science, 260:1937-1942 (1993). Recently, it has been shown that an
inhibitor of farnesyl-protein transferase blocks the growth of ras-
dependent tumors in nude mice (N.E. Kohl et al., Proc. Natl. Acad. Sci
U.S.A., 91:9141-9145 (1994) and induces regression of m~mmzlry 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 polyisoprenoids 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
loc~ tion in cultured cells. However, direct inhibition of ~arnesyl-
protein transferase would be more specific and attended by fewer side
..
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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
5 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 cont~ining
molecules that are related to the CAAX motif that is the signal for
protein prenylation. (Schaber et al., ibid; Reiss et. al., ibid; Reiss et al.,
PNAS, 88:732-736 (1991)). Such inhibitors may inhibit protein
prenylation while serving as alternate substrates for the farnesyl-protein
transferase enzyme, or may be purely competitive inhibitors (U.S.
Patent 5,141,851, University of Texas; N.E. Kohl et al., Science,
260:1934-1937 (1993); Graham, et al., J. Med. Chem., 37, 725 (1994)).
15 In 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
20 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 thereapy of
arteriosclerosis and diabetic disturbance of blood vessels (JP H7-
25 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-cont~ining inhibitors of farnesyl protein transferase have also
30 been disclosed (WO 95/09001 and EP 0 675 112 Al).
It is, therefore, an object of this invention to develop
peptidomimetic compounds that do not have a thiol moiety, and that will
inhibit farnesyl-protein transferase and thus, the post-trz~n~l~tional
farnesylation of proteins. It is a further object of this invention to
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-- 4 --
develop chemotherapeutic compositions cont~inin~ the compounds of
this invention and methods for producing the compounds of this
invention.
-5 SUMMARY OF THE INVENTION
The present invention comprises small molecule
- peptidomimetic amide-cont~inin~ compounds which inhibit the farnesyl-
protein trallsferase. The instant cornpounds lack a thiol moiety and thus
offer unique advantages in terms of improved pharmacokinetic behavior
10 in ~nim~l~, prevention of thiol-dependent chemical reactions, such as
rapid autoxidation and disulfide formation with endogenous thiols, and
reduced systemic toxicity. Further contained in this invention are
chemotherapeutic compositions cont~inin.~ these farnesyl transferase
inhibitors and methods for their production.
The compounds of this invention are illustrated by the
formula I:
( lR6)r !1 7~ C(R1~)2C(Rl1)2 /R3
V - Al(CRla2)nA2~CRla2)n -\W~!- (CRlb2~ A3 - (CR2 )--Y
o Rl2 R4
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DETAIL~D DESCRIPTION OF THE INVENTION
The compounds of this invention are useful in the inhibition
of farnesyl-protein transferase and the farnesylation of the oncogene
protein Ras. In a first embodiment of this invention, the inhibitors of
5 farnesyl-protein transferase are illustrated by the formula I:
( I 6)r ~I 7~ ~ (R )2C~R )2 ~R3
V - A1 (CR1 a2)nA2(CR 1 a2)n -\W~!- (CR1 b2~ A3 - (CR2 ~_y
O R12 R4
whereln:
Rla~ R1b and R2 are independently selected from:
a) hydrogen,
b) aryl, heterocycle, C3-Clo cycloaL~yl, C2-C6 alkenyl, C2-
C6 aL~ynyl, R80-, R9s(o)m-~ R8C(O)NR8-, CN, N02,
(R8)2N-C(NR8)-, R8C(O)-, R8OC(O)-, N3, -N(R8)2, or
R9OC(o)NR8-,
c) C1-C6 alkyl unsubstituted or substituted by aryl,
heterocyclic, C3-Clo cycloalkyl, C2-c6 alkenyl, C2-C6
alkynyl, R8O-, R9S(o)m-~ R8C(O)NR8-, CN, (R8)2N-
C(NR8)-, R8C(O)-, R8OC(O)-, N3, -N(R8)2, or
R9OC(o)-NR8-;
R3 and R4 are independently selected from F, Cl, Br, N(R8)2, CF3,
NO2, (R8)o-, (R9)S(o)m-~ (R8)C(O)NH-, H2N-
C(NH)-, (R8)C(O)-, (R8)OC(O)-, N3, CN,
CF3(CH2)nO-, (R9)OC(O)NR8-, Cl-C20 alkyl,
substituted or unsubstituted aryl and substituted or
unsubstituted heterocycle;
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R5 is selected from:
a) hydrogen,
b) unsubstituted or substituted aryl,
c) unsubstituted or substituted heterocyclic,
d) unsubstituted or substituted C3-Clo cycloaLkyl, and
e) Cl-C6 aLkyl substituted with hydrogen or a group
selected from unsubstituted or substituted aryl,
unsubstituted or substituted heterocyc}ic, unsubstituted or
substituted C3-C10 cycloalkyl, N(R8~2, CF3, N02, (R8)o-,
(R9)S(o)m-~ (R8)C(O)NH-, H2N-c(NH)-~ (R8)C~O)-,
(R8)OC(O)-, N3, CN (R9)OC(O)NR8-;
R6 is independently selected from:
a) hydrogen,
b) aryl, heterocycle, C3-C10 cycloalkyl, C2-C6 alkenyl, C2-
C6 aLkynyl, periluoroaLkyl, F, Cl, Br, R80-, R9S(o)m
R8C(O)NR8-, CN, NO2, R82N-C(NR8)-, R8C(O)-,
R80C(O)-, N3, -N(R8)2, or R9OC(o)NR8-, and
c) Cl-C6 alkyl unsubstituted or substituted by aryl,
heterocycle, C3-Clo cycloaLkyl, C2-c6 alkenyl, C2-C6
alkynyl, per~uoroaLkyl, F, Cl, Br, R80-, R9S~o)m-,
R8C(O)NH-, CN, H2N-C(NH)-, R8C(O)-, R8OC(O)-, N3,
-N(R8)2, or R80C(O)NH-;
25 R7 is selected from:
a) hydrogen,
b) C2-C6 aLkenyl, C2-C6 alkynyl, perfluoroalkyl, F, Cl, Br,
R80-, R9S(o)m-~ R8C(O)NR8-, CN, N02,
(R8)2N-C-(NR8)-, R8C(O)-, R8OC(O)-, N3, -N(R8)2, or
R9OC(o)NR8-, and
c~ Cl-C6 aLkyl unsu~stituted or substituted by perfluoroaLkyl,
~, Cl, Br, R8O-, R9S(O)m-, R8C(O)NR8-, CN, (R8)2N-
C(NR8)-, R8C(O)-, R80C(O)-, N3, -N(R8)2, or
R9OC(o)NR8-;
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R8 is independently selected from hydrogen, C1-C6 alkyl, benzyl and
aryl;
S R9 is independently selected from Cl-C6 aLkyl and aryl;
\f~ NR82
R10 and R11 are independently selected from: H; ~ or
Cl 5 alkyl, unbranched or branched, unsubstituted or
substituted with one or more of:
1) aryl,
2) heterocycle,
3) oR8,
4) SR9, SO2R9, or
5) ~f NR82
R12 is H, C1-Clo alkyl, substituted or unsubstituted aryl or C1-
Clo aLkyl which is substituted with a substituted or
unsubstituted aryl;
20 Al and A2 are independently selected from: abond, -CH=CH-, -C--C-,
-C(O)-, -C(O)NR8-,-NR8C(O)-, O, -N(R8)-,
-S(0)2N(R8)-, -N(R8)S(0)2-, or S(O)m;
A3 is selected from: -NR5- or a bond;
V is selected from:
a) hydrogen,
b) heterocycle,
. c) aryl,
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.
_
-- 8 -
d) Cl-C20 aLkyl wherein from O to 4 carbon atoms are
replaced with a a heteroatorn selected from 0, S, and N,
and
e) C2-C20 aL~enyl,
5 provided that V is not hydrogen if Al is S(O)m and V is not hydrogen
if Al is a bond, n is O and A2 is S(O)m;
W is a heterocycle;
10 Y is aryl or heteroaryl;
m is 0, 1 or 2;
nis 0, 1, 2, 3 or4;
pis 0, 1, 2, 3 or4;
r is O to 5, provided that r is O when V is hydrogen, and
tis Oor l;
or the ph~rmaceutically acceptable salts thereof.
A preferred embodirnent of the compounds of this
invention are illustrated by the formula Ia:
( lR6)r R7a
V ~ A1(cR1a2)nA2(cRla2~N \y
) - N A3- (CR2 )~
O Rl2 R4
wherein:
Rla and R2 are independently selected from: hydrogen or Cl-C6 aLkyl;
Rlb is independently selected from:
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a) hydrogen,
b) aryl, heterocycle, cycloaL~yl, R80-, -N(R8)2 or C2-C6
aL~enyl,
~ c) Cl-C6 alkyl unsubstituted or substituted by aryl,
S heterocycle, cycloaL~yl, alkenyl, R80-, or-N(R8)2;
R3 and R4 are independently selected from F, Cl, Br, N(R8)2, CF3,
NO2, (R8)o-, (R9)S(O)m-, (R8)C(O)NH-, H2N-
C(NH)-, (R8)C(O)-, (R8)OC(O)-, N3, CN,
(R9)OC(O)NR8-, Cl -C20 alkyl, substituted or
unsubstituted aryl and substituted or unsubstituted
heterocycle;
R5 is selected from:
a) hydrogen,
and
b) Cl-C6 alkyl substituted with hydrogen or a group
selected from unsubstituted or substituted aryl,
unsubstituted or substituted heterocyclic, unsubstituted or
substituted C3-Clo cycloaLlcyl, N(R8)2~ CF3, NO2, (Rg)O-,
(R9)S(o)m-~ (R8)C(O)N~-, H2N-C(NH)-, (R8)C(O)-,
(R8)OC(O)-, N3, CN (R9)OC(O)NR8-;
R6 is independently selected from:
a) hydrogen,
b) Cl-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, Cl-C6
perfluoroaL~yl, F, Cl, R80-, R8C(O)NR8-, CN, N02,
(~8)2N-C(NR8)-, R8C(O)-, RgOC(O)-, -N(R8)2, or
R9OC(o)NR8-, and
c) Cl-C6 alkyl substituted by Cl-C6 perfluoroaL~yl, R80-,
R8C(O)NR8-, (R8)2N-C(NR8)-, R8C(O)-, R80C(O)-,
-N(R8)2, or R9OC(o)NR8-;
R7a is hydrogen or methyl;
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- 10 -
R8 is independently selected from hydrogen, Cl-c6 aLkyl, benzyl and
aryl;
S R9 is independently selected from Cl-C6 aLkyl and aryl;
~ NP~82
R10 and Rll are independently selected from: H; O or
Cl 5 aLkyl, unbranched or branched, unsubstituted or
substituted with one or more of:
1) aryl,
2) heterocycle,
3) o:R-8,
4) SR9, SO2R9, or
5) NR82
O
R12 is H, Cl-Clo aLkyl and substituted or unsubstituted aryl;
A1 and A2 are independently selected from: a bond, -CH=CH-, -~C-,
-C(O)-, -C(O)NR8-, O, -N(R8)-, or S(O)m;
A3 is selected from: -NR5- or a bond;
V is selected from:
a) hydrogen,
b) heterocycle selected from pyrrolidinyl, imidazolyl,
pyridinyl, thiazolyl, pyridonyl, 2-oxopiperidinyl, indolyl,
quinolinyl, isoquinolinyl, and thienyl,
c) aryl,
d) Cl-C20 aLkyl wherein from 0 to 4 carbon atoms are
replaced with a a heteroatom selected from O, S, and N,
and
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.
.
- 11 -
e) C2-C20 alkenyl, and
provided that V is not hydrogen if Al is S(O)m and V is not hydrogen
if Al is a bond, n is 0 and A2 is S(O)m;
5 mis 0, lor2;
nis 0, 1, 2, 3 or4;
p is 0, 1, 2, 3 or 4; and
r is 0 to 5, provided that r is 0 when V is hydrogen;
10 or the pharmaceutically acceptable salts thereof.
A second preferred embodiment of the compounds of this
invention are illustrated by the formula Ib:
( l 6)r
V - A1(CR1a2)nA2(CR1a2)n -\W~!- (CR1b2~ A3 - (CR22)p~¦~
Ib o R12 R4
wherein:
Rla and R2 are independently selected from: hydrogen or Cl-C6 alkyl;
20 Rlb is independently selected from:
a) hydrogen,
b) aryl, heterocycle, cycloalkyl, R8O-, -N(R8)2 or C2-C6
alkenyl,
c) Cl-C6 alkyl unsubstituted or substituted by aryl,
- 25 heterocycle, cycloalkyl, alkenyl, R8O-, or-N(R8)2;
- R3 and R4 are independently selected from F, Cl, Br, N(R8)2, CF3,
N02, (R8)o-, (R9)S(o)m-~ (R8)C(O)NH-, H2N-
C(NH)-, (R8)C(O)-, (R8)OC(O)-, N3, CN,
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(R9)OC(O)NR8-, Cl-C20 alkyl, substituted or
unsubstituted aryl and substituted or unsubstituted
heterocycle;
5 R5 is selected from:
a) hydrogen,
and
b) Cl-C6 alkyl substituted with hydrogen or a group
selected from unsubstituted or substituted aryl,
unsubstituted or substituted heterocyclic, unsubstituted or
substituted C3-Clo cycloaL~cyl, N(R8)2, CF3, NO2, (R8)o-,
(R9)s(o)m-~ (R8)C(O)NH-, H2N-c(NH)-~ (R8)C(O)-,
(R8)0C(O)-, N3, CN (R9)OC(O)NR8-;
15 R6 is independently selected from:
a) hydrogen,
b) Cl-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, Cl-c6
perfluoroalkyl, F, Cl, R8O-, R8C(O)NR8-, CN, NO2,
(R8)2N-C(NR8)-, R8C(O)-, R8OC(O)-, -N(R8)2, or
R9OC(o)NR8-, and
c) Cl-C6 alkyl substituted by Cl-C6 perfluoroaLkyl, R80-,
R8C(O)NR8-, (R8)2N-C(N~,8), R8C(o) R80C(o)_
-N(R8)2, or R9OC(o)NR8-;
25 R7 is selected from: hydrogen and Cl-C6 alkyl;
R8 is independently selected from hydrogen, Cl-C6 aLkyl, benzyl aIld
aryl;
~0 Rg is independently selected from Cl-C6 aL~cyl and aryl;
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.
-
- 13 -
NR82
~ ;
R10 and Rll are independently selected from: H; O or
~ Cl 5 alkyl, unbranched or branched, unsubstituted or
substituted with one or more of:
1 ) aryl,
2) heterocycle,
3) oR8,
4) SR9, SO2R9, or
5) ~NR82
o
R12 is Cl-Clo alkyl and substituted or unsubstituted aryl;
Al and A2 are independently selected from: a bond, -CH=CH-, -C~C-,
-C(O)-, -C(O)NR8-, O, -N(R8)-, or S(O)m;
15 A3 is selected from: -NR5- or a bond;
V is selected from:
a) hydrogen,
b) heterocycle selected from pyrrolidinyl, imidazolyl,pyridinyl, thiazolyl, pyridonyl, 2-oxopiperidinyl, indolyl,
quinolinyl, isoquinolinyl, and thienyl,
c) aryl,
d) C l-C20 aL~yl wherein from 0 to 4 carbon atoms are
replaced with a a heteroatom selected from O, S, and N,
and
e) C2-C20 aL~enyl, and
provided that V is not hydrogen if Al is S(O)m and V is not hydrogen
if Al is a bond, n is 0 and A2 is S(O)m;
30 W is a heterocycle selected from pyrrolidinyl, pyridinyl, thiazolyl,
pyridonyl, 2-oxopiperidinyl, indolyl, quinolinyl, or isoquinolinyl;
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- 14 -
m is 0, l or 2;
nis 0, 1, 2, 3 or4;
pis 0, 1, 2, 3 or4;
r is 0 to 5, provided that r is 0 when ~,7 is hydrogen; and
tis 1;
or the pharmaceutically acceptable salts thereof.
In a more preferred embodiment of this invention, the
inhibitors of farnesyl-protein transferase are illustrated by the formula
Ic:
N ~ N ~ ~ ~
~¦~ (CRlb2)p - N r - (cR2, ~ ~
R6 ~ Rl2 R5 R4
lc
15 wherein:
Rlb is independently selected frorn:
a~ hydrogen,
b) aryl, heterocycle, cycloalkyl, RgO-, -N(R8)2 or C2-C6
alkenyl,
c) Cl-C6 aLkyl unsubstituted or substituted by aryl,
heterocycle, cycloaLkyl, alkenyl, R80-, or-N(R8)2;
R2 are independently selected from: hydrogen or Cl-C6 aLkyl;
R3 and R4 are independently selected from F, Cl, Br, N(R8~2, CF3,
N02, (R8)o-, (R9)S(o)m-~ (R8)C(O)NH-, ~I2N-
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C(NH)-, (R8)C(O)-, (R8)OC(O)-, N3, CN,
(R9)OC(O)NR8-, Cl-C20 alkyl, substituted or
unsubstituted aryl and substituted or unsubstituted
heterocycle;
s
R5 is selected from:
a) hydrogen,
and
b) Cl-C6 alkyl substituted with hydrogen or a group
selected from unsubstituted or substituted aryl,
unsubstituted or substituted heterocyclic, unsubstituted or
substituted C3-Clo cycloalkyl, N(R8)2, CF3, NO2, (R8)o-,
(R9)S(O)m-, (R8)C(O)NH-, H2N-C(NH)-, (R8)C(O)-,
(R8)OC(O)-, N3, CN (R9)OC(O)NR8-;
R6 is independently selected from:
a) hydrogen,
b) Cl-C6 aL~yl, C2-C6 aL~enyl, C2-C6 alkynyl, Cl-C6
perfluoroalkyl, F, Cl, R8O-, R8C(O)NR8-, CN, NO2,
(R8)2N-C(NR8)-, R8C(O)-, R8OC(O)-, -N(R8)2, or
R9OC(o)NR8-, and
c) Cl-C6 alkyl substituted by Cl-C6 perlluoroalkyl, R80-,
R8C(O)NR8-, (R8)2N-C(NR8)-, R8C(O)-, R80C(O)-,
-N(R8)2, or R9OC(o)NR8-;
R8 is independently selected from hydrogen, Cl-c6 alkyl, benzyl and
aryl;
R9 is independently selected from Cl-C6 alkyl and aryl;
N R82
Il ;
~R10 and Rll are independently selected from: H; O or
Cl 5 alkyl, unbranched or branched, unsubstituted or
substituted with one or more of:
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- 16 -
1 ) aryl,
2) heterocycle,
3) oR8,
4) SR9, SO2R9, or
5) ~ NR52
O
R12 is Cl-Clo alkyl and substituted or unsubstituted aryl;
mis 0,lor2;and
p is 0, 1, 2, 3 or 4;
or the pharmaceutically acceptable salts thereof.
In a second more preferred embodiment of this invention,
15 the inhibitors of farnesyl-protein transferase are illustrated by the
forrnula Id:
N~N C(Rl ~)2c(R 11~ 3
NC ~ R12 R5 R4
Id
wherein:
Rlb is independently selected from:
a) hydrogen,
b) aryl, heterocycle, cycloaLkyl, R80-, -N(R8)2 or C2-C6
alkenyl,
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W 097127853 PCT~US97/01455
c) C l-C6 alkyl unsubstituted or substituted by aryl,
heterocycle, cycloalkyl, alkenyl, R80-, or-N(R8)2;
~2 are independently selected from: hydrogen or Cl-C6 alkyl;
R3 and R4 are independently selected from F, Cl, Br, N(R8)2, CF3,
N02, (R8)o-, (R9)S(O)m-, (R8)C(O)N~I-, H2N-
C(NH)-, (R8)C(O)-, (R8)OC(O)-, N3, CN,
(R9)OC(O)NR8-, Cl-C20 alkyl, substituted or
unsubstituted aryl and substituted or unsubstituted
heterocycle;
R5 is selected from:
a) hydrogen,
and
b) Cl-C6 alkyl substituted with hydrogen or a group
selected from unsubstituted or substituted aryl,
unsubstituted or substituted heterocyclic, unsubstituted or
substituted C3-Clo cycloaLkyl, N(R8)2~ CF3, NO2, (R8)o-,
(R9)S(V)m-~ (R8)C(O)NH-, H2N-C(NH)-, (R8)C(O)-,
(R8)OC(O)-, N3, CN (R9)OC(O)NR8-;
R8 is independently selected from hydrogen, Cl-C6 alkyl, benzyl and
aryl;
R9 is independently selected from Cl-C6 aL~yl and aryl;
IR82
R10 and Rll are inde~endently selected from: H; ~ or
- Cl 5 alkyl, unbranched or branched, unsubstituted or
substituted with one or more of:
- 1 ) aryl,
2) heterocycle,
3) oR8,
CA 02243272 1998-07-15
W 097/27853 PCTrUS97/01455
- 18 -
4) SR9, SO2R9, or
5) \~f NR82
O
R12 is Cl-Clo aLkyl and substituted or unsubstituted aryl;
mis 0,lor2;and
pis 0, 1, 2, 3 or4;
or the pharmaceutically acceptable salts thereof.
Specific examples of the compounds of the invention are:
N-[ 1 -(4-cyanobenzyl)-5-imidazolylmethyl]-N-[2-((3-
chlorophenyl)arnino)ethyl~acetamide
c
NC ~<~N~H3c
or
N-[ 1-(4-cyanobenzyl)-5-irnidazolyImethyl]-N-(3-
phenylpropyl)acetamide
NC ~<~N~H3c
or the pharmaceutically acceptable salts thereof.
CA 02243272 1998-07-l~
W097/27853 PCTAJS97/0145S
.
- 19 -
The compounds of the present invention may have
asymmetric centers and occur as ~acemates, racemic mixtures, and as
individual diastereomers, with all possible isomers, including optical
isomers, being included in the present invention. When any variable
S (e.g. aryl, heterocycle, Rla, R2 etc.~ occurs more than one time in any
constituent, its definition on each occurence is independent at every
other occurence. Also, combinations of substituents/or variables are
permissible only if such combinations result in stable compounds.
As used herein, "alkyl" is intended to include both branched
and straight-chain saturated aliphatic hydrocarbon groups having the
specified number of carbon atoms; "alkoxy" represents an alkyl group
of indicated number of carbon atoms attached through an oxygen
bridge. "Halogen" or "halo" as used herein means fluoro, chloro,
bromo and iodo.
As used herein, "aryl" is intended to mean any stable
monocyclic or bicyclic carbon ring of up to 7 members in each ring,
wherein at least one ring is aromatic. Examples of such aryl elements
include phenyl, naphthyl, tetrahydronaphthyl, indanyl, biphenyl,
phenanthryl, anthryl or acenaphthyl.
The term heterocycle or heterocyclic, as used herein,
represents a stable S- to 7-membered monocyclic or stable 8- to l l-
membered bicyclic heterocyclic ring which is either saturated or
lln~tllrated, and which consists of carbon atoms and from one to four
heteroatoms selected from the group consisting of N, O, and S, and
including any bicyclic group in which any of the above-defined
heterocyclic rings is fused to a benzene ring. The heterocyclic ring may
be attached at any heteroatom or carbon atom which results in the
creation of a stable structure. Examples of such heterocyclic elements
include, but are not limited to, azepinyl, benzimidazolyl, benzisoxazolyl,
benzofurazanyl, benzopyranyl, benzothiopyranyl, benzofuryl,
benzothiazolyl, benzothienyl, benzoxazolyl, chromanyl, cinnolinyl,
dihydrobenzofuryl, dihydrobenzothienyl, dihydrobenzothiopyranyl,
dihydrobenzothiopyranyl sulfone, furyl, imidazolidinyl, imidazolinyl,
imidazolyl, indolinyl, indolyl, isochromanyl, isoindolinyl, isoquinolinyl,
CA 02243272 1998-07-15
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- 20 -
isothiazolidinyl, isothiazolyl, isothiazolidinyl, molpholinyl,
naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, 2-oxopiperazinyl, 2-
oxopiperdinyl, 2-oxopyrrolidinyl, piperidyl, piperazinyl, pyridyl,
pyrazinyl, pyrazolidinyl, pyrazolyl, pyridazinyl, pyrimidinyl,
5 pyrrolidinyl, pyrrolyl, quinazolinyl, quinolinyl, quinoxalinyl,
tetrahydrofuryl, tetrahydroisoquinolinyl, tetrahydroquinolinyl,
thiamorpholinyl, thiamorpholinyl sulfoxide, thiazolyl, thiazolinyl,
thienofuryl, thienothienyl, and thienyl.
As used herein, "heteroaryl" is intended to mean any stable
10 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,
15 benzofurazanyl, benzopyranyl, benzothiopyranyl, benzofuryl,
benzothiazolyl, benzothienyl, benzoxazolyl, chromanyl, cinnolinyl,
dihydrobenzofuryl, dihydrobenzothienyl, dihydrobenzothiopyranyl,
dihydrobenzothiopyranyl sulfone, furyl, imidazolyl, indolinyl, indolyl,
isochromanyl, isoindolinyl, isoquinolinyl, isothiazolyl, naphthyridinyl,
20 ox~ olyl, pyridyl, pyrazinyl, pyrazolyl, pyridazinyl, pyrimidinyl,
pyrrolyl, quinazolinyl, quinolinyl, c~uinoxalinyl,
tetrahydroisoquinolinyl, tetrahydroquinolinyl, thiazolyl, thienofuryl,
thienothienyl, and thienyl.
As used herein, the terms "substituted aryl", "substituted
25 heterocycle" and "substituted cycloalkyl" are intended to include the
cyclic group which is substituted with 1 or 2 substitutents selected from
the group which includes but is not limited to F, Cl, Br, CF3, NH2,
N(Cl-C6 alkyl)2, NO2, CN, (cl-c6 aLkyl)O-, -OH, (Cl-C6
aL~cyl)S(O)m-, (cl-c6 alkyl)C(O)NH-, H2N-C(NH)-, (Cl-C6
30 aLkyl)C(O)-, (Cl-C6 aL~cyl)OC(O)-, N3,(Cl-C6 alkyl)OC(O)NH- and
Cl-C20 aLkyl.
Lines drawn into the ring systems from substituents (such
as from R2, R3, R4 etc.) indicate that the indicated bond may be
attached to any of the substitutable ring carbon atoms.
CA 02243272 1998-07-l~
W O 97/27853 PCTrUS97/01455
.
Preferably, Rla, Rlb and R2 are independently selected
from: hydrogen, -N(R8)2~ R8~(O)NR8- or Cl-c6 alkyl unsubstituted
or substituted by -N(R8)2, R80- or R8C(O)NR8-.
Preferably, R3 and R4 are independently selected from:
5 hydrogen, perfluoroalkyl, F, Cl, Br, R8O-, R9s(o)m-~ CN, NO2,
R82N-C(NR8)-, R8C(O)-, R8OC(O)-, N3, -N(R8)2, or R9OC(o)NR8-
and C1-C6 aL~yl.
Preferably, R5 is selected from hydrogen or Cl-C6 alkyl
substituted with hydrogen, R9S(O)m-, CF3- or an unsubstituted or
10 substituted aryl group.
Preferably, R6 is selected from: hydrogen, perfluoroaL~yl,
F, Cl, Br, R8O-, R9s(o)m-~ CN, NO2, R82N-C(NR8~-, R8C(O)-,
R80C(O)-, N3, -N(R8)2, or R9OC(o)NR8- and Cl-C6 aL~yl.
Preferably, R7 is hydrogen.
Preferably, R8 is selected from ~I, Cl-C6 aL~yl and benzyl.
Preferably, R9 is selected from Cl-C6 aL~yl.
Preferably, R10 and Rl 1 is selected from H, Cl -C6 alkyl
and benzyl.
Preferably, R12 is selected from Cl-C6 aL~yl. More
preferably Rl 2 is methyl.
Preferably, Al and A2 are independently selected from: a
bond, -C(O)NR8-, -NR8C(O)-, O, -N(R8)-, -S(0)2N(R8)- and-
N(R8)S(0~2-
Preferably, V is selected from hydrogen, heterocycle and
aryl. Most preferably, V is phenyl.
Preferably, Y is selected from phenyl, furyl, thienyl and
pyridyl. Most preferably, Y is phenyl.
Preferably, n, p and r are independently 0, 1, or 2.
Preferably t is 1.
The pharrnaceutically 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,
CA 02243272 1998-07-15
W 097~78~3 PCT~US97/01455
- 22 -
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, maleic, hydroxymaleic,
phenylacetic, ~ t~mic, benzoic, salicylic, sulfanilic, 2-acetoxy-benzoic,
5 fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic,
isethionic, tril~uoroacetic and the like.
It is intended that the definition of any substituent or
variable (e.g., Rla, Z, n, etc.) at a particular location in a molecule be
independent of its definitions elsewhere in that molecule. Thus,
-N(R8)2 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
15 forth below, from readily available starting materials.
T~e ph~ ceutically 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
20 chromatography or by reacting the free base with stoichiometric
amounts or with an excess of the desired salt-forming inorganic or
organic acid in a suitable solvent or various combinations of solvents.
Reactions used to generate the compounds of this invention
are prepared by employing reactions as shown in Schemes 1-12, in
2~ addition to other standard manipulations such as ester hydrolysis,
cleavage of protecting groups, etc., as may be known in the literature or
exemplified in the experimental procedures. Substituents R and R CH2-
, as shown in the Schemes, represent the substitllçnts R8, R9 ~d others,
depending on the compound of the instant invention that is being
30 synthesized. The variable p' represents p-l.
These reactions may be employed in a linear sequence to
provide the compounds of the invention or they may be used to
synthesize fragrnents which are subsequently joined by the aLkylation
reactions described in the Schemes.
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W 097/27853 PCTrUS97tO1455
_
- 23 -
Synopsis of Schemes 1-12:
The requisite intermediates are in some cases commercially
available, or can be prepared according to literature procedures, for the
5 most part. Schemes 1-2 illustrates the synthesis of one of the preferred
embodiments of the instant invention, wherein the variable W is present
as a imidazolyl moiety that is substituted with a suitably substituted
benzyl group. Substituted protected imidazole alkanols II carl be
prepared by methods known in the art, such as those described by F.
Schneider, Z. Physiol. C~em., 3:206-210 (1961) and C.P. Stewart,
Biochem. Journal, 17:130-133(1923). Benzylation and deprotection of
the imidazole aLkanol provides intermediate III which can be oxidized to
the corresponding aldehyde IV.
The aldehyde whose synthesis is illustrated in Scheme 1
15 may be reacted with a suitably substituted ~ mine VI, which was
prepared from the ~niline V as shown in Scheme 2, to provide the
intermediate compound VII. Compound VII can be selectively N-
acylated under standard conditions, such as those illustrated, to provide
the instant compound VIII. The analogous reaction directed towards
20 compounds wherein A3 is a bond is illustrated in Scheme 2a.
Schemes 3-6 illustrate syntheses of suitably substituted
aldehydes useful in the syntheses of the instant compounds wherein the
variable W is present as a pyridyl moiety. Similar synthetic strategies
for preparing alkanols that incorporate other heterocyclic moieties for
25 variable W are also well known in the art.
The ~ mine VI can be reacted with a variety of other
aldehydes, such as IX, as shown in Scheme 7. The product X is first
acylated and then can be deprotected to give the instant compound XI.
The compound XI is isolated in the salt form, for example, as a
30 trifluoroacetate, hydrochloride or acetate salt, among others. As shown
in Scheme 8, Compound XI can further be selectively protected to
obtain XII which can subsequently be reductively alkylated with a
second aldehyde, such as XIII, to obtain XIV. Removal of the
-
CA 02243272 1998-07-15
WO 97127853 PCTrUS97/01455
- 24 -
protecting group, and conversion to cyclized products such as the
dihydroimidazole XV can be accomplished by literature procedures.
If the ~ mine VI is reductively aLkylated with an aldehyde
which also has a protected hydroxyl group, such as XVI in Scheme 9,
the product XVII can first be acylated and the protecting groups can be
subsec~uently removed to llnm~k the hydroxyl group (Schemes 9, 10).
The alcohol can be oxidized under standard conditions to e.g. an
aldehyde, which can then be reacted with a variety of organometallic
reagents such as Grignard reagents, to obtain secondary alcohols such as
10 XXI. In addition, the fully deprotected amino alcohol XXII can be
reductively alkylated (under conditions described previously~ with a
variety of aldehydes to obtain secondary amines, such as XXIII (Scheme
10), or tertiary amines.
The Boc protected amino alcohol XIX can also be utilized
15 to synthesize 2-aziridinylmethylamides such as XXIV (Scheme 11).
Treating XIX with l,l'-sulfonyldiimidazole and sodium hydride in a
solvent such as dimethylformz-micle leads to the formation of aziridine
XXIV. The aziridine may be reacted with a nucleophile, such as a thiol,
in the presence of base to yield the ring-opened product XXVI.
In addition, the ~ rnTne VI can be reacted with aldehydes
derived from amino acids such as O-alkylated tyrosines, according to
standard procedures, to obtain compounds such as XXXII, as shown in
~cheme 12. Intermediate XXXII is ~lrst acylated before it is further
elaborated. When R' is an aryl group, XXXIII can first be
25 hydrogenated to nnm~k the phenol, and the amine group deprotected
with acid to produce XXXIV. Alternatively, the arnine protecting
group in XXXIII can be removed, and O-aLkylated phenolic arnines such
as XXXV produced.
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- 25 -
SCHEME 1
(CR1b2)P~~cH20Hprot1x Et3N Prot 'N~/~
N N
H DMF 11
(CR1b2)p~-cH2oAc R6~\~EtOAc
Ac20. Py
prot1--N ~ N 2. N-deprotect
(CR1 b2)p.-CH20Ac (CR1 b2) -CH20H
R6~ THF, H20 6~
111
(CR1b ) .-CHO
S03 Py, Et3N >
DMSO
R6/~
IV
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_
- 26 -
SCHEME 2
H2N ~ HCI
V ~/~\
R3 R4
R10 H
\_N
R1~ )c
R11 0
R11
R1o~1 1
H N ~/~, N~
Vl /~\
R3 R4
~ (CR1b2)p'-CHO
1. aq. NaHCO3 2. N~
6 ~ Na(ACO)3BH
R
lV
1b2) .-cH2NHc(R1o)2c~R1 )2N~
R3 R4
R
Vll
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- 27 -
SCHEME 2 (continued)
1b2) . CH2NHC(R1~)2C(R11)2N~
R3 R4
R6~
Vll (Rl2c=o)2o
or
R12C=OCI
R112~o
(CR1 b2)p,-CH2NC(Rl~)2C(R 11 )2NH
> R3 R4
R
Vlll
CA 02243272 1998-07-15
W O 97127853 PCTrUS97101455
- 28 -
SCHEME 2a
R10,1 1
HCI H2N /~~3
R3 R4
(CRl b2)p,-CHO
1. aq. NaHCO3 2. N~
~ Na(AcOj3BH
r R6
~(CRlb2)p, CH2NHC(Rt~)2C(R11~2C~
> R3 R4
R6i~ (R 1 2C=0)20
or
R12C=OCI
R12~o
(CR1 b2)p.-CH2NC(Rl ~)2C(R 1 1 )2C~
R3 R4
R6/~
CA 02243272 1998-07-15
W O 97/27853 PCT~US97/01455
.
_
- 29 -
SCHEME 3
CH3 1 ) HNO2,Br2 ~C02CH3
~ 2) KMnO
H2N N~ 3) MeOH,H+ Br~N~
R6
~\~ MgCI R6
~ CO2CH3
Zncl2~Ni(~l2(ph3p)2 N
R6
NaBH4 (excess) ~ ,CH20H
SO3 Py, Et3N ~CHO
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PCTAUS97/01455
- 30 -
SCHEME 4
R6
~CO2CH3 ~\ N~
Zn, CuCN
R6 R6
NaBH4 ~j~ S03Py, Et3N
(excess)
~,CH20H DMSO ~CHO
R6 R6
Br~CO2CH3 ~\ MgCI
N Zncl2, NiC12(Ph3P)2 ~CO2CH3
R6 R6
NaBH4
(excess) ~CH20H Y 3, ~CHO
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- 31 -
S (~ EME S
CO2CH3
Br~1. LDA, CO2 Br~
N2. MeOH, H+ N
ZnCI2, NiC12(Ph3P)2 N
R6
NaBH4 (excess) ~2OH SO3Py, Et3N
DMSO
R6
CHO
CA02243272 1998-07-lF,
WO 97n7853 PCT/US97/01455
- 32 -
SCHEME 6
C02CH3
1. LDA, CO2 ~Br
2. (CH3~3SiCHN2
R6 ~/\ Br R6 ~
N ~CO2CH3
Zn, NiC12(Ph3P)2 ~1
R6 1~
excess NaBH4 l~ SO3 Py, Et3N
~CH20-l DMSO
R6 ~
N~3~CH~
-
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- 33 -
~C~ME 7
Boc NH IX
R10,1 1
H N~/~N~ Boc NH CHO
IX ~/~\ NaBH(OAc)3
R3 R4 Et3N, CICH2CH2CI
Vl
Boc NH~NHC(R1~)2C(Rll)2NH (Rl2C-0)20
NHBoc / \
X R3/~\R4 R C=OCI
R1 ~o
Boc NH rNC(Rl~)2C(Rl1~2~ CF CO H
NHBoc ~'/.?,\J CH2CI2
R3 R4
R1 ~o
NH2 rNC(R1~)2C(R1l)2NH
NH2 ~'/ \J
Xl R3 R4
CA 02243272 1998-07-15
WO 97127853 PCT~US97/014S5
- 34 -
SCHEME 8
N H ~ NC(R )2C(R )2 ~ Boc2O
NH2 ~/~\ CH2CI2
Xt R3 R4
~f ~3,CHX10
BocNH rNC(R1~)2C(R11)2NH
~~ ~ NaBH(OAc)3
NH2 ~/~\ Et3N, CtCH2CH2CI
Xl I R3 R4
R12 0
BocNH~NC(R )2C(R )2~CF3CO2H, CH2CI2;
NH
~/ XIV R3 R4
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WO 97/27853 PCTrUS97/01455
SCHEME 8 (continued)
R12 o
NH~ NC(R1~)2C(R11)2NH ~NC
NH R3 \R4
R12~o
~ NC(Rl~)2C(Rl1)2NH
N~,N~ /~,\
¢~ XV R3 R4
~3
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- 36 -
SCHEME 9
R10,1 1 NaBH(OAc)3
H N~/~N~ Et3N, CICH2CH2CI
Vl '' 4 BnOl
BocNH CHO
XVI
BnO~ NHC(Rl~)2C(Rll)2NH1. (R12C=0)20
NHBoc / \ R12C=OCI
XVI I R3 R4 2 . (Boc)2O
R12~fO
Boc
BnO ~NC(Rl~)2C(Rll)2N~l 20/o Pd(oH)2~ H2
NHBoc /~\ CH3Co2H
XVI 1 I R3 R4
R12~o
HO NC(R )2C(R )2N CICOCOCI
~,~:~ DMSO CH2CI2
NHBoc l~ (C2Hs)3N
Yl~
~'~ R3 R4
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SCHEME 9 (CONTINUED)
R12~0 1. R'MgX
~~ NG~~32C~ )2N (C2H5)2o
H NHBoc \~ CH2CI2
XX R3 R4
R12~o
HO>~ NC(R1 ~)2C(R1 1 )2N H
R' NH2 ~/~\
XXI R3 R4
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- 38 -
SCHEME 10
HO ~ NC(R )2C(Rl1)2N CF3CO2H
NHBoc ~1~¦ CH2CI2
XIX R3 R4
R1 ~o
HO ~ NC(Rl~)2C(Rl l)2NH R'CHO
NH2 \~3 NaBH(OAc)3
/~\ 4 Clc H2CH2
XXI I R3 R
R1~o
HO~ NC(R1~)2C(Rl l)2NH
NH
R'CH2 R3 R4
XXIII
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- 39 -
SCHEME 1 1
R12 0 H H
~ Boc N=\ ~N
HO ~--NC(Rl~)2C(R11)2N o2
NHBoc / \
XIX '' 4NaH, Dl\/F 0~C
R12~o
<~ NC(R1~)2C(R1l)2N R'SH
N \~ (C2Hs)3
Boc XXIv ~ 4 CH30H
R12~o
Boc
R'S ~ NC(R1~)2C(R1132N HCI, EtOAc
NHBoc
XXV R3 R4
R12 0
R'S ~ NC(R1~)2C(R11)2N~3
NH2 /~\
XXVI R3 R4
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- 40 -
SCHEME 12
H,~3 1) Boc20, Ko2C~3 ,~
H2NCO2H 2) CH2N2, EtOAc CO2CH3
XXVII
XXVIII
HO,~
LiAlH4 ~J~J R'CH2X
THF J~ Cs2CO3
0-20~C BocNH CH2OH DMF
XXIX
R'C H20 ~ ~ R'C H20
pyridine SO3~ ,~
DMSO
BocNH Cl 120H (c2H5)3N BocNH CHO
XXX XXXi
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- 41 -
SCHEME 12 (continued~
R'CH20~ R10,1 1
"~ + HCI H2N /--H~3
BocNH CHO /~\
XXXI Vl
NaBH(OAc)3
CICH2CH2CI
NHC( 1~)2C(R1 l)2NH
XXXII HCI
(R12C=0)20
or
R12C=OCI
'f
yNC(R )2C(R )2N~
NHBoc ~
R3 R4
XXXIII
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- 42 -
SCHEME 12 ~continued)
~, R12~0
R'CH20 NC(R1~)2C(Rl 1)2NH
NH2 /~\
/i' XXXV R3 R4
HCI
ETOAc
~NC(R )2C(R ~2N~
NHBoc ~ 4
XXXIII
1) 20% Pd(OH)2
CH30H, CH3CO2H
2) HCI, EtOAc
~ R12 0
HO~ NC(R1~)2C(R11)2NH
NH2 /~\
XXXIV R3 R4
- -
CA 02243272 1998-07-1~
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.
- - 43 -
The instant compounds are useful as ph~rm~reutical agents
for m~mm~ , especially for humans. These compounds may be
~Amini.~tered to patients for use in the treatrnent of cancer. Examples of
the type of cancer which may be treated wi~ the compounds of this
5 invention include, but are not lin~ited 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 formation (i.e.,
neurofibromen (NF-l), neu, scr, abl, lck, fyn) or by other mech~ni~m~.
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 angiogenisis, thereby
affecting the growth of tumors (J. Rak et al. Cancer Research, 55:4575-
4580 (1995)). Such anti-angiogenisis properties of the instant
compounds may also be useful in the treatment of certain forms of
blindness related to retinal vasc~ r 7~tion.
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 ~mini~tration of an effective amount of the compounds of the
invention to a m~mm~l in need of such treatment. For example, a
component of NF-l 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 translllmin~l coronary
angioplasty by inhibiting neointim~l 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.
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American Journal of Pathology, 142:1051-1060 (1993) and B. Cowley,
Jr. et al.FASEB Journal, 2:A3160 (1988)).
The compounds of this invention may be ~1rninistered to
m~mm~l~, preferably humans, either alone or, preferably, in
combination with ph~ eeutically acceptable carriers or diluents,
optionally with known adjuvants, such as alum, in a ph~ eutical
composition, according to standard pharmaceutical practice. The
compounds can be ~ ini~tered orally or parenterally, including the
intravenous, intramuscular, intraperitoneal, subcutaneous, rectal and
topical routes of ~clministration.
For oral use of a chemotherapeutic compound according to
this invention, the selected compound may be ~-lmini~tered, for
example, in the form of tablets or capsules, or as an aqueous solution or
suspension. In the case of tablets for oral use, carriers which are
commonly used include lactose and corn starch, and lubricating agents,
such as magnesiurn stearate, are commonly added. For oral
~lmini~tration in capsule form, useful diluents include lactose and dried
corn starch. VVhen aqueous suspensions are required for oral use, the
active ingredient is combined with emulsifying and suspending agents.
If desired, certain sweetening and/or flavoring agents may be added.
For intramuscular, intraperitoneal, subcutaneous and intravenous use,
sterile solutions of the active ingredient are usually prepared, and the pH
of the solutions should be suitably adjusted and buffered. For
intravenous use, the total concentration of solutes should be controlled
in order to render the preparation isotonic.
As used herein, the term "composition" is intended to
encompass a product comprising the specified ingredients in the specific
amounts, as well as any product which results, directly or indirectly,
from combination of the specific ingredients in the specified amounts.
The present invention also encompasses a pharmaceutical
composition useful in the treatment of cancer, comprising the
~(1mini~tration of a therapeutically effective amount of the compounds
of this invention, with or without pharmaceutically acceptable carriers
or diluents. Suitable compositions of this invention include aqueous
~,.
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solutions comprising compounds of this invention and pharmacolo-
gically acceptable carriers, e.g., saline, at a pH level, e.g., 7.4. The
solutions may be introduced into a patient's intramuscular blood-stream
by local bolus injection.
S When a compound according to this invention is
~f1mini~tered into a human sub~iect, the daily dosage will normally be
determined by the prescribing physician with the dosage generally
varying according to the age, weight, 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 ~flmini~tered to a m~mm~l undergoing treatment for
cancer. Aflmini.~tration 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 O.S mg/kg of body weight to about 40 mg/kg of body weight
per day.
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 transferase (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 in~t~nt invention. After the assay mixtures are
incubated for an sufficient period of time, well known in the art, to
allow the ~PTase to farnesylate the substrate, the chemical content of
the assay mixtures may be determined by well known
immunological, radiochemical or chromatographic techniques.
Because the compounds of the instant invention are selective
inhibitors of ~;PTase, absence or quantitative reduction of the amount
3~ of substrate in the assay mixture without the compound of the instant
invention relative to the presence of the unchanged substrate in the
assay cont~inin~; the instant compound is indicative of the presence of
F~PTase in the composition to be tested.
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- 46 -
It would be readily apparent to one of ordinary skill in the
art that such an assay as described above would be usefill in identifying
tissue samples which contain farnesyI-protein transferase and
quantitating the enzyme. Thus, potent inhibitor compounds of the
S instant invention may be used in an active site titration assay to
determine the quantity of enzyme in the sample. ~ series of samples
composed of aliquots of a tissue extract cont~ining an unknown arnount
of farnesyl-protein transferase, an excess amount of a known substrate
of FPTase (for example a tetrapeptide having a cysteine at the amine
10 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 suffilciently potent
inhibitor (i.e., one that has a Ki subst~nti~lly smaller than the
concentration of enzyme in the assay vessel) required to inhibit the
15 enzymatic activity of the sample by 50% is approximately equal to half
of the concentration of the enzyme in that particular sample.
EXAMPLES
Fx~rnples provided are intended to assist in a further
unders~ncling of the invention. Particular materials employed, species
and conditions are intended to be further illustrative of the invention
and not limitative of the reasonable scope thereof.
EXAMPLE 1
N-[l -(4-cyanobenzyl)-5-imidazolylmethyl]-N-[2-((3-
chlorophenyl)amino)ethyllacetamide dihydrochloride (1)
30 Step 1: Preparation of l-triphenylmethyl-4-(hydroxymethyl)-
imidazole (2)
To a solution of 4-(hydroxymethyl)imidazole
hydrochloride (35 g) in 250 mL of dry DMF at room temperature was
added triethylamine (90.6 mL). A white solid precipitated from the
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.
-
- 47 -
solution. Chlorotriphenylmethane (76.1 g) in 500 mL of DMF was
added dropwise. The reaction mixture was stirred for 20 hours, poured
over ice, filtered, and washed with ice water. The resulting product was
slurried with cold dioxane, filtered, and dried in vacuo to provide 2 as a
white solid which was sufficiently pure for use in the next step.
~tep 2: Preparation of 1-triphenylmethyl-4-(acetoxymethyl)-
imidazole (3)
Alcohol 2 (prepared above) was suspended in 500 mL of
pyridine. Acetic anhydride (74 mL) was added dropwise, and the
reaction was stirred for 48 hours during which it became homogeneous.
The solution was poured into 2 L of EtOAc, washed with water (3 x 1
L), 5% aq. HCl soln. (2 x 1 L), sat. aq. NaHCO3, and brine, then dried
(Na2SO4), filtered, and concentrated in vacuo to provide the crude
product. The acetate 3 was isolated as a white powder which was
sufficiently pure for use in the next step.
Step 3: Preparation of 1-(4-cyanobenzyl)-5-(acetoxymethyl)-
imidazole hydrobromide (4)
A solution of 3 (85.8 g) and a-bromo-p-tolunitrile (50.1 g)
in 500 mL of EtOAc was stirred at 60 ~C for 20 hours, during which a
pale yellow precipitate formed. The reaction was cooled to room
temperature and filtered to provide the solid imidazolium bromide salt.
The filtrate was concentrated in vacuo to a volume 200 mL, reheated at
60 ~C for two hours, cooled to room temperature, and filtered again.
The filtrate was concentrated in vacuo to a volume 100 mL, reheated at
60 ~C for another two hours, cooled to room temperature, and
concentrated in vacuo to provide a pale yellow solid. All of the solid
material was combined, dissolved in 500 mL of methanol, and warmed
to 60 ~C. After two hours, the solution was reconcentrated in vacuo to
provide a white solid which was triturated with hexane to remove
soluble materials. Removal of residual solvents in vacuo provided the
titled product hydrobromide as a white solid which was used in the next
step without further purification.
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Step 4: Preparation of 1-(4-cyanobenzyl)-5-(hydroxymethyl)-
imidazole (5)
To a solution of the acetate 4 (50.4 g) in 1.5 L of 3:1
5 THF/water at 0 ~C was added lithium hydroxide monohydrate (18.9 g).
After one hour, the reaction was concentrated in vacuo, diluted with
EtOAc (3 L), and washed with water, sat. aq. NaHCO3 and brine. The
solution was then dried (Na2SO4), filtered, and concentrated in vacuo to
- provide the crude product as a pale yellow fluffy solid which was
10 sufficiently pure for use in the next step without further purification.
,~tep 5: Preparation of 1-(4-cyanobenzyl)-5-imidazole-
carboxaldehyde (6)
To a solution of ~e alcohol 5 (21.5 g) in 500 mL of DMSO
15 at room temperature was added triethylamine (56 mL), then S O3-
pyridine complex (40.5 g). After 45 minutes, the reaction was poured
into 2.5 L of EtOAc, washed with water (4 x 1 L) and brine, dried
(Na2SO4), ~lltered, and concentrated in vacuo to provide the aldehyde 6
as a white powder which was suf~lciently pure for use in the next step
20 without further purification.
Step 6: Preparation of N-(2-aminoethyl)-3-chloroaniline
}~ydrochloride (7)
To a solution of 3-chloro~niline (30 mL) in 500 mL of
25 dichloromethane at 0 ~C was added dropwise a solution of 4 N HCl in
1,4-dioxane (80 mT ). I~e solution was warmed to room temperature,
then concentrated to dryness in vacuo to provide a white powder. A
mixture of this powder with 2-oxazolidinone (24.6 g) was heated under
nitrogen atmosphere at 160 ~C for 10 hours, during which the solids
30 melted, and gas evolution was observed. The reaction was allowed to
cool, forming the titled compound 7 as a pale brown solid.
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Step 7: Preparation of N-[1-(4-cyanobenzyl)-5-imidazolylmethyl~-
N'-(3-chlorophenyl)ethylene~ mine (S)
The amine hydrochloride 7 (978 mg) was partitioned
between dilute aqueous NaHCO3 solution and methylene chloride. The
aqueous layer was washed with three portions of CH2Cl2, and the
combined organics were dried (Na2SO4), filtered, and concentrated in
vacuo to provide the free a~nine. To a solution of the amine in 11 mL
of 1,2-dichloroethane at 0 ~C was added 4A powdered molecular sieves
(2 g), followed by sodium triacetoxyborohydride (3.04 g). The
aldehyde 6 (1.21 g) was added, and the reaction was stirred at 0 ~C.
After lS hours, the reaction was poured into EtOAc, washed with
saturated aqueous NaHCO3, and the aqueous layer was extracted with
EtOAc. The combined organics were washed with brine, dried
(Na2SO4), ~lltered, and concentrated in vacuo. The resulting product
was taken up in 60 mT of S:l benzene:CH2Cl2, and propyl~rnine (10
mL) was added. The reaction was stirred for 12 hours, then
concentrated in vacuo, and puri~led by silica gel chromatography (5%
MeOH/CHCl3) to provide the titled compound 8 as a white foam
Step 8: Preparation of N-[1-(4-cyanobenzyl)-5-imidazolylmethyl]-
N- [2-((3 -chlorophenyl)amino)ethyl] acetamide
dihydrochloride (1)
To a solution of the ~lt~lnine 8 (150 mg) in 2.5 mL of
CH2Cl2 was added triethylamine (0.057 mL). The solution was cooled
to 0 ~C, and acetic anyhydride (0.019 mL) was added. The reaction was
stirred overnight, allowing it to gradually warm to room temperature.
The mixture was poured into EtOAc and washed with sat. aq. NaHCO3
and brine, dried (Na2SO4), filtered, and concentrated in vacuo. This
material was purified by silica gel chromatography (2.5-5%
MeOH/CH2Cl2), taken up in CH2Cl2 and treated with 1 M HCl/ether
solution, and concentrated in vacuo. The product hydrochloride 1 was
isolated as a white solid.
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FAB mass spectrum m/e 408 (M+l).
Analysis calculated for C22H22ClNsO ~ 2.00 HCl - 0.50 H20:
C, 53.95; H, 5.14; N, 14.30;
Found: C, 53.93; H, 5.42; N, 13.42.
EX~MPLE 2
N- [1 -(4-cyanobenzyl)-S-imidazolylmethyl] -N-(3 -
phenylpropyl)acetamide hydrochloride (9)
St~p 1: Preparation of N-[1-(4-cyanobenzyl)-5-imidazolylmethyl~-
N-(3-phenylpropyl~amine (10)
To a solution of 3-phenylpropylamine (0.202 mL, 1.42
mmol) in 5 mL of 1,2-dichloroethane at 0 ~C was added 4A powdered
lS molecular sieves (0.38 g), followed by sodium triacetoxyborohydride
(301 mg, 1.42 mmol g). The aldehyde 6 from Step 5 of Fx~ le 1
(200 mg, 0.947 mmol) was added, and the reaction was allowed to
warm to room temperature. After 2 days, the reaction was poured into
EtOAc, washed with sat. aq. NaHCO3, and the aqueous layer was
extracted with EtOAc. The combined organics were washed with brine,
dried (Na2SO4), filtered, and concentrated in vacuo. The resulting
product was taken up in 12 mL of 20% CH2Cl2/ propylamine, stirred
for 12 hours, concentrated in vacuo, and purified by preparative HPLC.
Thus, the product was taken up in water/MeOH solution and was
injected directly onto a Delta-Pal~ (C-18, 100A, 15 mm, 40 mm x 100
mrn) prep HPLC column using a gradient with 0.1% trifluoroacetic
acid/water and 0. l ~o trifluoroacetic acid/acetonitrile as solvents. A
portion of the pure fractions was then partitioned between me~lene
chloride and water, and the organic phase was dried (Na2SO4), filtered,
and concentrated in vacuo to provide the titled product 10 as a white
solid.
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Step 2: Preparation of N-[1-(4-cyanobenzyl)-5-imidazolylmethyl~-
N-(3-phenylpropyl)acetamide hydrochloride (9)
To a solution of the amine 10 from Step 1 (41 mg, 0.125
mmol) in 2 mL of CH2Cl2 was added triethyl~mine (0.035 mL, 0.250
rnmol). The solution was cooled to 0 ~C, and acetic anyhydride (0.012
mL, 0.125 mmol) was added. After 1 hour, the mixture was poured
into EtOAc and washed with sat. aq. NaHCO3 and brine, dried
(Na2SO4), filtered, and concentrated in vacuo. This material was
purified by silica gel chromatography (70% acetone/hexane), taken up
in CH2Cl2 and treated with 1 M HCl/ether solution, and concentrated in
vacuo. The titled product hydrochloride 9 was isolated as a white solid.
FAB mass spectrum m/e 373 (M~l).
Analysis calculated for C23H24N4O ~ 1.00 HCl - 0.90 H2O:
C, 64.98; H, 6.35; N, 13.18;
Found: C, 65.10; H, 6.32; N, 12.82.
F.XAMPLE 3
In vitro inhibition of ras farnesyl transferase
Assays of farnesyl-protein transferase. Partially purified
bovine FPTase and Ras peptides (Ras-CVLS, Ras-CVIM and Ras-CAIL)
were prepared as described by Schaber et a1., J. Biol. Chem. 265: 14701-
14704 (1990), Pompliano, et al., Biochemistry 31:3800 (1992) and
Gibbs et al., PNAS U.S.A. 86:6630-6634 (1989), respectively. Bovine
FPTase was assayed in a volume of 100 ~l contz-ining 100 mM N-(2-
hydroxy ethyl) piperazine-N'-(2-ethane sulfonic acid) (HEPES), pH 7.4,
S rnM MgCl2, 5 mM dithiothreitol (DTT), 100 mM [3H]-farnesyl
diphosphate ([3H]-FPP; 740 CBq/mmol, New Fngl~ntl Nuclear), 650 nM
Ras-CVLS and 10 ,ug/ml FPTase at 31~C for 60 min. Reactions were
initiated with FPTase and stopped with 1 ml of 1.0 M HCL in ethanol.
Precipitates were collected onto filter-mats using a TomTec Mach II cell
~ harvestor, washed with 100% ethanol, dried and counted in an LKB ~-
plate counter. The assay was linear with respect to both substrates,
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FPTase levels and time, less than 10% of the [3H3-FPP was lltili7ecl
during the reaction period. Purified compounds were dissolved in
100% dimethyl sulfoxide (DMSO) and were diluted 20-fold into the
assay. Pelcell~age inhibition is measured by the arnount of
5 incorporation of radioactivity in the presence of the test compound
when compared to the amount of incorporation in the absence of the test
compound.
Human FPTase was prepared as described by Omer et al.,
Biochemistry 32:5167-5176 (1993). Human FPTase activity was
10 assayed as described above with the exception that 0.1% (w/v)
polyethylene glycol 20,000, 10 ,llM ZnCl2 and 100 nM Ras-CVIM were
added to the reaction mixture. Reactions were performed for 30 min.,
stopped with 100 ,ul of 30% (v/v) trichloroacetic acid (TCA) in ethanol
and processed as described above for the bovine enzyme.
The compound of the instant invention described
hereinabove in F.x~mrle 1 was tested for inhibitory activity against
human FPTase by the assay described above and was found to have ICso
of< 10,uM.
F.X~MPLE 4
In vivo 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 labelled in 3 ml methionine-~ree DMEM supple-
meted with 10% regular DMEM, 2% fetal bovine serum and 400
mCi[35S~methionine ~1000 Ci/mrnol). After an additional 20 hours, the
cells are lysed in 1 ml lysis buffer ~% NP40/20 mM HEPES, pH 7.5/5
mM MgC12/lmM DTT/10 mg/ml aprotinen/2 mg/ml leupeptin/2 mg/ml
antipain/0.5 mM PMSF) and the lysates cleared by centrifugation at
100,000 x g for 45 min. Aliquots of lysates con~:lining equal numbers
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of acid-precipitable counts are bought to 1 ml with IP buffer (lysis
buffer lacking DTT) and immunoprecipitated with the ras-specific
monoclonal antibody Y13-259 (Furth, M.E. et aL, J. Virol. 43:294-30~,
~ (1982)). Following a 2 hour antibody incubation at 4~C, 200 ml of a
5 25% suspension of protein A-Sepharose coated with rabbit anti rat IgG
is added for 45 min. The immllnoprecipitates are washed four times
with IP buffer (20 nM HEPES, pH 7.5/1 mM EDTA/1% Triton X-
100Ø5% deoxycholate/0.1%/SDS/0.1 M NaCl) boiled in SDS-PAGE
sample buffer and loaded on 13% acrylamide gels. When the dye front
10 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 5
~n v~o ~rowth inhibition assay
To deterrnine 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
in.ct~nt compounds for Ras-induced cell transformation.
Rat 1 cells transformed with either v-ras, v-raf, or v-mos
are seeded at a density of 1 x 104 cells per plate (35 mm in diameter) in
a 0.3% top agarose layer in medium A (Dulbecco's modified Eagle's
medium supplemented with 10% fetal bovine serum) over a bottom
agarose layer (0.6%). Both layers contain 0.1% methanol or an
appropriate concentration of the instant compound (dissolved in
methanol at 1000 times the final concentration used in the assay). The
cells are fed twice weekly with 0.5 ml of medium A cont~ining 0.1%
methanol or the concentration of the instant compound.
Photomicrographs are taken 16 days after the cultures are seeded and
comparisons are made.
