Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THE INVENTION
~NHIBITORS OF FARNESYL-PROTEIN TRANSFERASE
S BACKGROUND OF THE ~VENTTON
The present invention relates to compounds which
inhibit farnesyl protein transferase, a protein which is implicated in the
oncogenic pathway mediated by Ras. The Ras proteins (Ha-Ras, Ki4a-
Ras, Ki4b-Ra.s and N-Ras) are part of a signalling pathway that links
cell surface growth factor receptors to nuclear signal.s 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 termin~ted by the intrinsic
GTPase activity of Ra,s, which returns the protein to its inactive GDP
bound form (D.R. Lowy and D.M. Willumsen, Ann. Rev. Biochem.
62:~51-~91 (1993)). Mutated ras genes (Ha-ras, Ki4a-)as, 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 ~re involved with Ras membrane localization, and all
3 modifications occur at the C-terminus of ~as. The Ras C-terminu.s
contain,s a sequence motif termed a "CAAX" or "Cy.s-Aaal-Aaa2-Xaa"
box (Cys is cy~steine, Aaa is an aliphatic amino acid, the Xaa is any
- 30 amino acid) (Willumsen et al., Natu) ~ 310:5~3-5~6 (19~4)). Depend-
ing on the specific .sequence, this motif serves as a signal .sequence for
- the enzymes farnesyl-protein transfera.se 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.,
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WO 97/36876 PCT/US97/~6257
Ann. Pev. Bi~chen~ 355-386 (1992); W.R. Schafer and J. Rine,
Ann. Rel~. Genetics 30:209-237 (1992)). Ras proteins are known to
undergo post-translational farnesylation. Other farnesylated proteins
include the Ras-related GTP-binding proteins such as Rho, fungal
5 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. James, et al., have also suggested that there are
farnesylated proteins of unknown ~structure and function in addition
10 to those listed above.
lnhibition 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
15 selectively block the processing of the Ras oncoprotein intracellularly
(N.E. Kohl et al., Science, 260:1934-1937 (1993) and G.L. James e~ al.,
Science, 260:1937-1942 (1993). Recently, it has been shown that an
inhibitor of farnesyl-protein transferase blocks the growth of ~as-
dependent tumors in nude mice (N.E. Kohl et al., Proc. Natl. Acad.
20 Sci U.S.A., 91:9141-9145 (1994) and induces regression of mammary
and salivary carcinomas in ras transgenic mice (N.E. Kohl et al..
Nature Medicine, 1 :792-797 (1995).
Indirect inhibition of farnesyl-protein transfera~se in l'iVO
has been demonstrated with lovastatin (Merck & Co., Rahway, NJ)
25 and compactin (Hancock et al., ibid; Casey et al., ibid; Schafer et al.,
Science 245:379 (l9~S9)). The.se 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
30 CAAX box with a farnesyl group (Reiss et al., Cell 62:~ g (1990);
Schaber et al., J. Biol. Chem., 2~5:14701-14704 (1990); Schafer et al.,
- Science, 249: 1133- 1139 (1990); Manne eF al., Pro( . Natl. Acad. Sci
USA, 87:7541-7545 (1990)). Inhibition of farnesyl pyropho~phate
biosynthesis by inhibiting HMG-CoA reductase blocks Ras membrane
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Iocalization in cultured cells. However, direct inhibition of farnesyl-
protein tran~sferase would be more specific, and thus preferable.
Inhibitor.s 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 i~ related to the
protein ~ubstrates (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., ihid; Reiss et. al., ihid; Reiss
0 et al., PNAS, 88:732-736 (1991)). Such inhibitors may inhibit protein
prenylation while serving as alternate substrates for the farnesyl-protein
tran,sferase enzyme, or may be purely competitive inhibitors (U.S.
Patent 5,141,~51, University of Texas; N.E. Kohl et al., Science,
260:1934-1937 (1993); Graham, et al., J. Med. Chem., 37, 725 (1994)).
It has recently been reported that FPT-ase inhibitor,s
also inhibit the proliferation of vascular smooth muscle cells and are
therefore useful in the prevention and treatment of arteriosclerosi,s and
diabetic disturbance of blood ves,sels (JP H7-112930).
It has recently been di,sclosed that certain tricyclic
20 compounds which optionally incorporate a piperidine moiety are
inhibitors of FPTase (WO 95/10514, WO 95/1051~ and WO 95/10516).
Imidazole-containing inhibitors of farne~yl protein transferase have also
been disclosed (WO 95/09001 and EP 0 675 112 Al).
25 SUMMARY OF THE INVENTION
The present invention addresses a compound of formula I:
V A1(CRla2) A2(CR'b2)n ~(W)~ (CR 2t~A - (CR 2)p
or a pharmaceutically acceptable ~alt thereof, wherein:
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Rla, R1b and R2 are independently selected from the
group consisting of: hydrogen? aryl, substituted aryl, heterocyclyl,
C3-Clo cycloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, R~O-, R9S(o)m
wherein m is 0, 1 or 2, R~C(O)NR8-, CN, NO2, (R8)2NC(NR8)-,
R8C(O)-, R80C(O)-, N3, -N(R8)2, R90C(o)NR8- and Cl-C6 alkyl,
unsubstituted or substituted by 1-3 groups .selected from the group
consisting of: halo, aryl, heterocyclyl, C3-clo cycloalkyl, C2-C6
alkenyl, C2-C6 alkynyl, R8O-, R9S(o)m-~ R8C(O)NR8-, CN,
(R8)2NC(NR8)-, R8C(O)-, R8OC(O)-, N3, -N(R~)2 and
1 0 R9OC(o)NR8-;
R3 and R4 are independently selected from the group
consisting of: H, F, Cl, Br, -NR82, CF3, NO2, R8O-, R9S(~)m-~
CF3(CH2)n-O-, R8C(O)NH-, H2NC(NH)-, R8C(O)-, R8OC(O)-, N3,
CN, R9OC(o)NR8-, C1-C20 alkyl, substituted or unsubstituted aryl and
,substituted or unsubstituted heterocyclyl;
A3 is selected from: --C----C , --R8C=CR8--
-C(O)-, aryl, heteroaryl or a bond;
provided that when A3 is heteroaryl, attachment of A3 the remainder of
the molecule is through substitutable heteroaryl ring carbon,s;
X represents aryl or heteroaryl;
provided that when X is heteroaryl, attachment of X the remainder of
the molecule is through substitutable heteroaryl ring carbons;
R6 is independently selected from the group consisting of:
hydrogen, aryl, heterocyclyl, C3-Clo cycloalkyl, C2-C6 alkenyl,
C2-C6 alkynyl, Cl-6 perfluoroalkyl, F, Cl, Br, R~O-, R9S(o)m-~
R8C(O)NR8-, CN, NO2, (R8)2Nc(NR8)-~ R8C(O)-, R8OC(O)-, N3,
-N(R8)2, R9OC(o)NR8- and Cl-C6 alkyl unsubstituted or substituted
by 1-3 group~s selected from: aryl, heterocyclyl, C3-Clo cycloalkyl,
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C2-C6 alkenyl, C2-C6 alkynyl, perfluoroalkyl, F, Cl, Br, R~O-,
R9S(o)m-~ R8C(O)NR~-, CN, (R~)2NC(NR~)-, R~C(O)-, R~OC(O)-,
N3, -N(R~)2 and R9OC(o)NR~-;
R7 is independently selected from the group consisting of:
hydrogen, aryl, heterocyclyl, C3-C1o cycloalkyl, C2-c6 alkenyl, C2-C6
alkynyl, C1 6 perfluoroalkyl, F, Cl, Br, R9O-, R9S(o)m-, R~C(O)NR~,
CN, NO2, (R8)2Nc(NR8)-~ R~C(O)-, R~OC(O)-, N3, -N(R8)2,
R9OC(o)NR8- and Cl-C6 alkyl unsub,stituted or substituted by 1-3
groups .selected from: aryl, heterocyclyl, C3-Clo cycloalkyl, C2-C6
alkenyl, C2-C6 alkynyl, perfluoroalkyl, F, Cl, Br, R8O-, R9S(o)m-~
R8C(O)NR8-, CN, (R8)2Nc(NR~ R8C(O)-, R~OC(O)-, N3, -N(R~)2
and R9OC(o)NR8-;
each R~ is independently selected from hydrogen, Cl-C6
alkyl, aryl and aralkyl;
each R9 is independently ~selected from Cl-C6 alkyl and
aryl;
Al and A2 are independently selected from the group
consisting of: a bond, -CH=CH-, -C_C-, -C(O)-, -C(O)NR8-,
-NR8C(O)-, -O-, -N(R8)-, -s(o)2N(R~ -N(R~)s(o)2-~ and S(O)m;
V is selected from the group consisting of: hydrogen,
heterocyclyl, aryl, Cl-C20 alkyl wherein from 0 to 4 carbon atoms are
replaced with a heteroatom ,selected from O, S, and N, and C2-c2o
alkenyl,
provided that V is not hydrogen if Al is S(O)m and V is not hydrogen
if Al isabond,n is0andA2isS(O)m;
provided that when V is heterocyclyl, attachment of V to R6 and to A
is through a substitutable ring carbon;
W represents heterocyclyl;
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each n and p independently represents 0, 1, 2t 3 or 4;
r i,s 0 to 5, provided that r is 0 when V is hydrogen, and
tis 1.
DETAILED DESCRIPTION OF THE INVENTION
The compounds of this invention are useful in the
inhibition of farne,syl-protein transferase and the farnesylation of the
oncogene protein Ras, and thus are useful for the treatment of cancer.
The compound~s of the present invention may have
asymmetric centers and occur as racemates, racemic mixtures and as
individual diastereomers, with all possible isomers, including optical
isomers, being included in the present invention.
When any variable (e.g. aryl, heterocycle, Rl, R2 etc.)
occurs more than one time in any constituent, each definition is
independent.
The term "alkyl" and the alkyl portion of alkoxy, aralkyl
and similar terms, is intended to include branched and straight-chain
saturated aliphatic hydrocarbon groups having the specified number
of carbon atoms, or 1-6 carbon atoms if unspecified. Cycloalkyl means
1-2 carbocyclic rings which are saturated and contain from 3-10 atom.s.
"Halogen" or "halo" as used herein means fluoro, chloro,
bromo and iodo.
As used herein, "aryl" and the aryl portion of aralkyl, are
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. A preferred
aralkyl group is benzyl.
The terms heterocyclyl, heterocycle and heterocyclic,
as used herein, mean a 5- to 7-membered monocyclic or ~s- to 11-
- membered bicyclic heterocyclic ring,s, either saturated or unsaturated,
aromatic, partially aromatic or non-aromatic, and which con~sist of
carbon atoms and from one to four heteroatoms selected from the group
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con.sisting of N, O, and S. Thus, it includes any bicyclic group in which
any of the above-defined heterocyclic rings i,s fu,sed to a benzene ring.
The ring or ring system may be attached at any heteroatom or carbon
atom which re.sults in a stable structure. lt optionally contains 1-3
S carbonyl groups. Example,s of such heterocycles include, but are not
limited to, azepinyl, benzimidazolyl, benzisoxazolyl, benzofurazanyl,
benzopyranyl, benzothiopyranyl, benzofuryl, benzothiazolyl,
benzothienyl, benzoxazolyl, chromanyl, cinnolinyl, dihydrobenzofuryl,
dihydrobenzothienyl, dihydrobenzothiopyranyl,
10 dihydrobenzothiopyranyl sulfone, furyl, imidazolidinyl, imidazolinyl,
imidazolyl, indolinyl, indolyl, isochromanyl, isoindolinyl, isoquinolinyl,
i~sothiazolidinyl, isothiazolyl, isothiazolidinyl, morpholinyl,
naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, 2-
oxopyrrolidinyl, pyridyl, pyrazinyl, pyrazolidinyl, pyrazolyl,
15 pyridazinyl, pyrimidinyl, pyrrolidinyl, pyrrolyl, quinazolinyl,
quinolinyl, quinoxalinyl, tetrahydrofuryl, tetrahydroisoquinolinyl,
tetrahydroquinolinyl, thiamorpholinyl, thiamorpholinyl sulfoxide,
thiazolyl, thiazolinyl, thienofuryl, thienothienyl and thienyl.
"Heteroaryl" is a subset of heterocyclic as defined above,
20 and means a monocyclic or bicyclic ring system, with 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 heteroatom~s selected from the
group consisting of N, O and S. Examples include benzimidazolyl,
benzisoxazolyl, benzofurazanyl, benzopyranyl, benzothiopyranyl,
25 benzofuryl, benzothiazolyl, benzothienyl, benzoxazolyl, chromanyl,
cinnolinyl, dihydrobenzofuryl, dihydrobenzothienyl,
dihydrobenzothiopyranyl, dihydrobenzothiopyranyl sulfone, furyl,
imidazolyl, indolinyl, indolyl, isochromanyl, isoindolinyl, isoquinolinyl,
isothiazolyl, naphthyridinyl, oxadiazolyl, pyridyl, pyrazinyl, pyrazolyl,
30 pyridazinyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolinyl,
quinoxalinyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, thiazolyl,
- thienofuryl, thienothienyl and thienyl.
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Lines drawn into ring sylstem.s from sub~stituents indicate
that the bond may be attached to any of the substitutable ring carbon
atoms.
Sub.stituted alkyl, substituted aryl, substituted heterocyclyl
and substituted cycloalkyl mean alkyl, aryl, heterocyclyl and
cycloalkyl groups, respectively, having from 1-3 substituents which are
selected from: halo, aryl, heterocyclyl, C3 1() cycloalkyl, C2 6 alkenyl,
C2 6 alkynyl, RXO-, R9S(o)m-~ RXC(O)NRx-, CN, (RX)2Nc(NRx)
RXC(O)-, RXOC(O)-, N3, -N(Rx)2 and R~OC(O)NRX-. When for
example, a substituted alkyl group i.s substituted with a "substituted aryl
group", the aryl portion is substituted with 1-3 groups as defined above.
Preferably 1-2 groups are present on substituted alkyl,
Isubstituted aryl, substituted heterocyclyl and substituted cycloalkyl,
which are selected from: halo, aryl, R8O-, CN, RXC(O)- and -N(RX)2.
Preferably, Rla ,R1b and R2 are independently selected
from: hydrogen, -N(R8)2, R8C(O)NR8- or unsubstituted or
substituted C1-C6 alkyl wherein the substituent on the substituted Cl-C6
alkyl is selected from un.substituted or substituted aryl, -N(R~)2, RfsO-
and R8C(o)NR8-
Preferably, R3 and R4 are selected from: hydrogen, Cl-C6
alkyl, Br, Cl, F, R8O-, and CF3.
In a preferred group of compounds, A3 represents
-C--C , -CR8=CR~s-, -C(O)- or a bond. A particularly preferred
group of compounds within this subset includes compounds of formula I
2~ wherein A3 represents -C--C-- or a bond.
Another preferred group of compounds includes the
compounds of formula I wherein A3 represents aryl or heteroaryl.
Preferably R6 represents CN.
Preferably, R7represents hydrogen, unsubstituted or
substituted Cl-C6 alkyl.
Preferably, RX represent.s H or Cl ~ alkyl, and R9 i.s C
- alkyl.
Preferably, Al and A2 are independently selected from:
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a bond, -C(O)NRX-, -NR~C(O)-, -O-, -N(RX)-, -S(O)2N(RX)- and-
N(Rg)S(0)2--
- Preferably, V is selected from hydrogen, heterocyclyl and
aryl. More preferably V is phenyl.
Preferably, W is heterocyclyl selected from imidazolinyl,
imidazolyl, oxazolyl, pyrazolyl, pyyrolidinyl, thiazolyl and pyridyl.
More preferably, W i.s selected from imidazolyl and pyridyl.
Preferably X represents aryl. In particular, X can
represent phenyl.
Preferably, m is 0 or 2.
Preferably n and p are 0, 1, 2 or 3.
A subset of compounds of the invention is represented by
forrnula Ia:
6 R7
(R )r
V - A1 (C R 1 a2)nA2(C R 1 b2)~3 N ~R3
(CR22)p--A3- (CR22)p X
\ 4
15 wherein:
R3, R4, A3, R~, R9, X, m, n, p and r are as originally
defined;
each R 1 a and R2 is independently selected from hydrogen
and Cl -C6 alkyl;
each Rlb is independently selected from: hydrogen, aryl,
heterocyclyl, C3 1() cycloalkyl, C2-~ alkenyl, R~O-, -N(R8)2 and Cl-C6
alkyl unsubstituted or substituted by aryl, heterocyclyl, cycloalkyl,
alkenyl,
RgO- and -N(RX)2;
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-10-
R6 is independently selected from: hydrogen, Cl-C6 alkyl,
C2-C6 alkenyl, C2-c6 alkynyl, Cl-C6 perfluoroalkyl, F, Cl, R80-,
R~C(O)NR~-, CN, NO2, (R~s)2N-c(NR~ R~sC(O)-, R~OC(O)-,
-N(Rg)2, or R9OC(o)NR8-, and Cl-C6 alkyl substituted by Cl-C6
5 perfluoroalkyl, R80-, R8C(O)NR8-, (R8)2N-C(NR8)-, R8C(O)-,
R8OC(O)-, -N(R8)2 and R9OC(o)NR8-;
R7 repre.sents H or Cl-6 alkyl;
A 1 and A2 are independently selected from: a bond,
-CH=CH-, -C_C-, -C(O)-, -C(O)NR2~-, O, -N(R8)- and S(O)m;
and V is selected from: hydrogen; aryl; heterocyclyl
selected from pyrrolidinyl, imidazolyl, pyridinyl, thiazolyl, indolyl,
~uinolinyl, isoquinolinyl and thienyl; Cl-C20 alkyl wherein from 0 to 4
carbon atoms are replaced with a a heteroatom selected from O, S, and
N, and C2-C20 alkenyl, provided that V is not hydrogen if Al is S(O)m
and V is not hydrogen if Al is a bond and A2 is S(O)m;
provided that when V is heterocycle, attachment of V to R6 and to Al is
through a substitutable ring carbon.
A second subset of compounds of the present invention is
represented by formula Ib:
(R6) /~7~ / R3
V-A1(CR1a2)nA2(CR1b2)n-\W~-(CR22)p-R3C=CR8~CR22),~X
Ib
wherein:
Rla Rlb, R2, Al, A2, R3, R4, R6, RX, R9, X, m, n, p and r
are as originally defined;
R7 is selected from: hydrogen and Cl-C6 alkyl;
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V is selected from: hydrogen, heterocyclyl ~elected from
pyrrolidinyl, imidazolyl, pyridinyl, thiazolyl, indolyl, quinolinyl,
isoquinolinyl and thienyl; aryl; Cl-c2o alkyl wherein from 0 to 4
carbon atom.s are replaced with a heteroatom selected from O, S, and N,
S and C2-C20 alkenyl,
provided that V is not hydrogen if Al is S(O)m and V is not hydrogen
if A 1 is a bond, n i~s 0 and A2 is S(O)m;
provided that when V is heterocycle, attachment of V to R~ and to Al is
10 through a substitutable ring carbon;
and
W represents heterocyclyl selected from pyrrolidinyl,
imidazolyl, pyridinyl, thiazolyl, indolyl, quinolinyl and isoquinolinyl.
A third subset of compounds of the present invention is
repre~ented by formula Ic:
(R6) (17~ ~R3
V-A1(CR1a2)nA2(CR1b2)n\W~~(CR22)pC_C--(CR2z~X
Ic R4
wherein:
Rl~ Rlb R2, Al, A2, R3, R4, R6, RX, R9, X, m, n, p and r
are as originally defined;
R7 is selected from: hydrogen and Cl-C6 alkyl;
V is selected from: hydrogen, heterocyclyl ,selected from
pyrrolidinyl, imidazolyl, pyridinyl, thiazolyl, indolyl, quinolinyl,
isoquinolinyl, and thienyl, aryl, Cl-C20 alkyl wherein from 0 to 4
carbon atoms are replaced with a heteroatom selected from O, S, and N,
and C2-C20 alkenyl,
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provided that V is not hydrogen if Al is S(O)m and V is not hydrogen
if Al i~sabond,n isOandA2isS(O)m;
provided that when V is heterocycle, attachment of V to R~ and to Al is
5 through a substitutable ring carbon;
and
W represents heterocyclyl selected from pyrrolidinyl,
imidazolyl, pyridinyl, thiazolyl, indolyl, quinolinyl and i.soquinolinyl.
A fourth embodiment of the invention is described in
accordance with formula Id:
H
)~N
,=~N~\J ~3
~¦~ (CR 2)p-A - (CR 2)p X~
D6
n Id
wherein:
each R2 is independently selected from hydrogen and
Cl-C6 alkyl;
R3, R4, A3, RX, R9, X, m and p are as originally
defined;
and R6 is selected from the group consisting of: hydrogen,
Cl-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, Cl-C6 perfluoroalkyl, F,
Cl, R~O-, R8C(O)NR8-, CN, NO2, (R8)2N-C(NR8)-, R8C(O)-,
25 R8OC(O)-, -N(R8)2, or R9OC(o)NR~- and Cl-C6 alkyl substituted by
Cl-C6 perfluoroalkyl, R80-, R8C(O)NR~-, (R8)2N-C(NR8)-, R8C(O),
R8OC(O)-, -N(R~)2 or R9OC(o)NRX-.
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-13-
A fifth sub.set of compounds of the invention is represented
by formula le:
H
)~N
(C R22)p A3 - (C R22)--X
NC
le
wherein:
X and A3 are as originally defined;
each R2 is independently selected from: hydrogen and
Cl-C6 alkyl;
R3 and R4 are independently ,selected from H, 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)0C(O)-, N3, CN, (R9)OC(O)NR8-, Cl-C20 alkyl,
substituted or unsub.stituted aryl, and substituted or unsubstituted
heterocyclyl;
and R~, R9, m and p are a.s originally defined.
Specific examples of compound.s of the invention are:
NC~ C--C~
<'~
N
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W 097/36876
-14-
NC ~ CH--CH~
N
NC ~ C--C
N
~\N ~ C H = C H ~ ~
NHo Cl
NC ~N~C _C
N
NC~3~N
NC ~ N~
N
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- 1 5 -
~>~N~? CH2-C--C
N
NC ~C--C
~\N~
S N
~_N CN
N
OH
N
' ~
N c'(3J ~3
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-16-
and the pharmaceutically acceptable salts and isomers thereof.
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, maleic, hydroxymaleic,
phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxy-benzoic,
fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic,
isethionic, trifluoroacetic and the like.
The pharmaceutically acceptable salts of the compounds
of this invention can be synthesized from the compounds of this
invention which contain a basic moiety by conventional chemical
methods. Generally, the salts are prepared either by ion exchange
chromatography or by reacting the free base with stoichiometric
amounts or with an excess of the desired salt-forming inorganic or
organic acid in a suitable ,solvent or variou,s combinations of solvents.
Reactions used to generate the compounds of this
invention are prepared by employing reactions as shown in the Schemes,
in addition to other standard manipulations such as ester hydrolysis,
cleavage of protecting groups, etc., as may be known in the literature
or exemplified in the experimental procedures. Substituents R and
R CH2-, as shown in the Schemes, represent the substituents R8, R9 and
others, depending on the compound of the instant invention that is being
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 u,sed to
synthesize fragments which are subsequently joined by the alkylation
reactions described in the Schemes.
Svnopsis of Schemes
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The requisite intermediates are in ~some cases commercially
available, or can be prepared according to literature procedures.
Schemes 1-2 illustrate the synthesi.s of one of the preferred embod~-
ment.s of the instant invention, wherein the variable W is present as an
imidazolyl moiety that is sub,stituted with a suitably sub,stituted benzyl
group. Substituted protected imidazoles can be prepared by method.s
such a,s those de.scribed by F. Schneider, Z. Physiol. Chem., 3:206-210
(1961) and C.P. Stewart, Biochem. Journal, 17:130-133(1923).
Benzylation and deprotection of the imidazole alkanol provide.s
intermediate III which can be oxidized to the corresponding aldehyde
IV. Also, while X is shown as a phenyl ring, other aryl and heteroaryl
groups can be substituted therein without departing from the invention.
The a~dehyde whose synthesis is illustrated in Scheme
1 may be reacted with ~ suitably substituted aralkyne, to provide
the intermediate compound V. Compound V can be selectively
hydrogenated across the unsaturated bond under standard conditions,
such as those illustrated, to provide Compound VI.
Schemes 3- 10 illustrate syntheses of suitably substituted
aldehydes u,seful in the syntheses of the instant compounds wherein
the variable W is a pyridyl moiety. Similar synthetic strategies for
preparing alkanols that incorporate other heterocyclic moieties for
variable W can be discerned from the teachings herein.
Generally the aldehyde i,s reacted with an appropriately
substituted aralkyne using n-BuLi, after which the triple bond can be
reduced. As shown in Schemes 2, 4, 6 and ~s reduction of the alkyne
triple bond using Pd/BaSO4 Iproduces the Z-olefin isomer almost
exclusively. By substituting sodium bi,~(2-methoxyethoxy)aluminum
hydride (RED-AL) in toluene, one can readily obtain the E-allylic
alcohol from propargylic alkynes used in the present invention.
In the preparation methods described herein,
reactive groups may remain blocked until the final product is prepared,
- essentially in protected form, after which a final deprotection step is
conducted. These blocking groups are readily removable i.e., they can
be removed, if desired, by procedures which will not cause cleavage or
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- 1 X -
other disruption of the remaining portions of the molecule. Such
procedures include chemical and enzymatic hydrolysis, treatment with
chemical reducing or oxidizing agents under mild conditions, treatment
with fluoride ion, treatment with a transition metal catalyst and a
5 nucleophile and catalytic hydrogenation.
Examples of suitable hydroxyl protecting groups are:
t-butylmethoxyphenylsilyl, t-butoxydiphenylsilyl, trimethylsilyl,
triethylsilyl, o-nitrobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl,
benzyloxycarbonyl, t-butyloxycarbonyl, 2,2,2-trichloroethyloxy-
10 carbonyl and allyloxycarbonyl. Preferred hydroxyl protecting groupsare trimethylsilyl and triethylsilyl.
Examples of suitable carboxyl protecting groups are:
benzhydryl, o-nitrobenzyl, p-nitrobenzyl, 2-naphthylmethyl, allyl,
2-chloroallyl, benzyl, 2,2,2-trichloroethyl, trimethylsilyl,
15 t-butyldimethylsilyl, t-butyldiphenylsilyl, 2-(trimethylsilyl)ethyl,
phenacyl, p-methoxybenzyl, acetonyl, p-methoxyphenyl,
4-pyridylmethyl and t-butyl. A preferred-carboxyl protecting group is
p-nitrobenzyl.
Many other suitable hydroxyl and carboxyl protecting
20 groups are known in the art. See, e.g., T.W. Greene, Protective Groups
in Or~anic Synthesis, John Wiley & Sons, Inc., 1981 (Chapters 2 and 5).
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19
SCHFME 1
~(cR22)Pl-cH2oH Prot1X Et3N Prot 'N~/~>
H DMF
lla
rBr
(C R22)p -C H20Ac R 6~EtOAc
AC20, Py ,=1=~
Prot1~N~ N 2. N-deprotect
CR2 ) -CH OAC N~(CR 2)p-CH20H
N~ LiOH
R6/~ THF, H20 ~
111
N,~(CR 2)p,-CHO HO r/R3
~N ~/ (CR22)p.-CH-C_C~_~
SO3Py, Et3N ~ n-BuLi, THF ~ > R4
DMSO~ H
IV R6
V
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SCHEME 2
R3
)p.-CH-C_C{;~
~N R4
R6 H2, Pd/C
V
MeOH, EtOAc
H2, Pd/BaSO4
MeOH, EtOAc HO R3
HO 3,~cR22)p-cH
~$CR22)p~-cH-CH=c~_;~ N
~N R4
(predominantly R6/~
~, Z isomer) Vlb
R6/~
Vla
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SCHEME 3
CH3 1) HN~2~Br2 CO2CH3
~ 2) KMnO
H2N N 3) MeOH,H+ Br N
R6
r ~\MgCI R6
~ ~,C02CH3
ZnCI2,NiCI2(Ph3P)2 N
R6
NaBH4 (excess) ~,CH20H
R6
SO3 Py Et3N ~CHO
OH R3
n-BuLi,THF R6
H--C--C{ ~ ~)~CH
R4
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SCHEME 4
OH R3
H2, Pd/BaSO4
MeOH, EtOAc
H2, Pd/C
~" MeOH, EtOAc
OH R3
R6 ~CH-CH=CH~
(predominantlyZ isomer) ~ ~CH~ R4
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SCHEME 5 R6
1. EtOC(=O)CI
~,CO2CH3 ~Z\ 9C CN ~C02CH3
N 3. S, xylene, heat N
R6 R6
NaBH4 ~ SO3Py, Et3N ~
(excess) ~"CH20H DMSO ~CHO
R6 R6
Br CO2CH3[~\ MgCI [~j
ZnCI2, Nicl2(ph3p)2 ~CO2CH3
R6 R6
NaBH4 ¦ SO3Py, Et3N
~CH20H ~ ~CHO
(excess) N DMSO NJJ
N-BuLi, THF ll f, OH
H = ~R~
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SCHEME 6
R6
OH
~,CH
N
H2, Pd/BaSO4 H2, Pd/C
MeOH, EtOAc MeOH, EtOAc
¢~3,CH-CH=CH~ 3,OH
(predominantly Z isomer)
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SCHEME 7
CO2C H3
Br~ 1. LDA, CO2 Br~
N 2. MeOH, H+ N
r
ZnC12, NiC12(Ph3P)2 N
R6
NaBH4 (excess) ~ ( ~20H S03Py, Et3N
DMSO
R6
R~
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SCHEME 8
R6
R4
N
H2, Pd/BaSO4 H2, Pd/C
MeOH, EtOAc MeOH, EtOAc
R6 R6
~'
N N
(predominantly Z isomer)
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SCHEME 9
C02C H3
1. LDA, CO2 [~Br
2. (CH3)3sicHN2
R6 ~Br R6 ~
~, co2CH3
Zn, NiC12(Ph3P)2 N~
R6 ~
excess NaBH4 ~ SO3 Py, Et3N
N ~CH20H DMSO
~,
R6 ~ R6 ~ OH
~CHO n-BuLi, THF N~ h R3
R3 R4
14
-
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SCHEME 10
R6 ~q
OH
~- = 7
H2, Pd/C R4
MeOH, EtOAc
R6 ~
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SCHEME 1 1
~/
BocN H ~
R4
BocNH CHO
n-BuLi, THF
BocN H
/=~ R CF3CO2H
BocNH~ ~ R4 CH2CI2
OH
NH2
R3 Boc20
NH2/~ R4 CH2CI2
OH
BocNH (R )r~ CHO
NH2~ R4 NaBH(OAc)3
OH Et3N, CICH2CH2CI
BocN H
(R )r~--CH2 ~ ~:~R3
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SCHEME 12
BocN H
~ R CF3CO2H
(R )~ CH 'NH~ R4 NaHCO3
NH~ ~R
(R )r~--CH2 ~ R4 AgCN
(R )r~ CH/ \~ 4
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SCHEME 13
H ~ R3
Me><O~ ~
Me N~\cHo R4
Boc n-BuLi, TH F
>< ~ R3 t-Bu(Me)2Si-CI
, ~R4 imidazole, DMF
Boc
OH
Me>< ~ R3 TsOH, H20
Boc R benzene
O-SiMe2(t-Bu)
HO
~ R CICOCOCI
BocNH~ R4 DMSO CH2C12
O-SiMe2(t-Bu) (c2H5)3N
O H
R3 1. R'MgX
BocNH--~R4 2. TFA
O-SiMe2(t-Bu) CH2CI2
HO R'
~ ~/ R
H2N~ R4
OH
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SCHEME 14
HO
~ R CF3CO2H
BocNH~ R4 CH2C12
O-SiMe2(t-Bu)
HO
~ R3 R'CHO
H2N~ R4 NaBH(OAc)3
OH CICH2CH2CI
HO
R'CH2~N A,~ R4
OH
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SCHEME 15
HO 3 N=~ ~= N
=~ R ~ ' S ~
BocNH~ ~ R4 ~2 ,,
O-SiMe2(t-Bu) NaH, DMF 0~C
Boc--N1 ~=~ R3 R'SH
4 (C2H5)3N ~,
R CH3oH
O-SiMe2(t-Bu)
R'S
~ R CF3CO2H
BocNH '\~ R4 CH2C12
O-SiMe2(t-Bu)
R'S ~ R3
H2N~ R4
OH
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SCHEME 16
HO,~1) Boc20, K2C~3 HO~
~/ THF-H20
2) CH2N2, EtOAc J~
H2NC02H BocNH CO2CH3
HO,,~
LiAlH4 ,b~l R'CH2X
THF l Cs2CO3
0-20~C BocNH CH2OH DMF
R'C H20 R'C H20
)~ pyridine SO
DMSO
BocNH CH20H (c2H5)3N BocNH~CHO
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SCHEME 16 (cont.)
R'CH ;~
BocNH CHO n-BuLi, THF
R'CH20~
BocNH J~ _ lc/R3
HO R4
HCI / \ BF3-0Et2, EtSH
ETOAc / \ CH2CI2
R'C H20~3 HO~
H2NJ~ ~R H2NJ~
The instant compound.s are useful in the treatment of
cancer. Cancers which may be treated with the compounds of this
5 invention include, but are not limited to, colorectal carcinoma,
exocrine pancreatic carcinoma, myeloid leukemia~ and neurological
tumor.s. Such tumors may arise by mutations in the ra~ genes
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themselves, mutations in the proteins that can regulate Ras activity (i.e.,
neurofibromin (NF-l), neu, scr, abl, lck, fyn) or by other mechani~sms.
The compounds of the instant invention inhibit farnesyl-
protein transferase and farnesylation of the oncogene protein Ras.
5 The instant compounds may also inhibit tumor angiogenesis, thereby
affecting the growth of tumors (J. Rak et al. Cancer Researc~h, 55:4575-
4580 (1995)). Such anti-angiogenic properties of the instant compounds
may also be useful in the treatment of certain forms of blindness related
to retinal vascularization.
The compounds of this invention are also useful for
inhibiting other diseases where Ras proteins are aberrantly activated a,s
a result of oncogenic mutation in other genes (i.e., the Ras gene itself i.s
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 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 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. Natu~ e
medicine, I :541 -545(1995).
The instant compounds may also be useful in the treatment
and prevention of polycystic kidney disease (D.L. Schaffner et al.
Ame~ican Journal of Pathology, 142:1051-1060 (1993) and B. Cowley,
Jr. et al.FASFB Journal, 2:A3160 (19~s~)).
The instant compounds may also be useful for the treatment
of fungal infections.
The compounds of this invention may be administered to
m~mm~ls, preferably humans, either alone or, preferably, in combina-
- tion with pharmaceutically acceptable carriers or diluents, in the form
of a pharmaceutical composition, which is comprised of a compound of
formula I in combination with a pharmaceutically acceptable carrier.
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The compounds can be administered orally, topically, rectally, vaginally
transderrnally or parenterally, including the intravenous, intramuscular,
intraperitoneal and ,subcutaneous routes of administration.
For oral use, the compound is administered, for example,
5 in the form of tablets or capsules, or as a solution or .suspen,sion. In the
case of tablets for oral use, carriers which are commonly used include
lactose and corn ,starch; lubricating agents, such as magnesium stearate,
are commonly added. For oral administration in capsule form, diluents
also include lactose and dried corn starch. When a4ueous suspensions
10 are required for oral use, the active ingredient is combined with
emulsifying and suspending agents. If de,sired, certain sweetening
and/or flavoring agents may be added. For intramuscular, intraperi-
toneal, subcutaneous and intravenous use, sterile solutions of the active
ingredient are usually prepared, the pH of the solution is suitably
15 adjusted and the product is buffered. For intravenous use, the total
concentration is controlled to render the preparation substantially
isotonic.
As used herein, the term "composition" i.s intended to
encompass a product comprising the specified ingredients in the specific
20 amounts, as well as any product which results, directly or indirectly,
from combination of the specific ingredients in the specified amount.s.
The compound,s of the instant invention may also be
co-~lmini~tered in therapeutic composition~s that also contain other
well known therapeutic agents that are selected for their particular
25 usefulness against the condition that is being treated. For example,
the instant compound.s may be useful in combination with known anti-
cancer and cytotoxic agents. Similarly, the instant compounds may be
u.seful in combination with agents that are effective in the treatment and
prevention of NF-I, restinosis, polycystic kidney disease, infection.s of
30 hepatitis delta and related viruses and fungal infections.
If formulated a~s a fixed dose, such combination products
- employ a compound of this invention substantially within the dosage
range described below and other pharmaceutically active agent(s~
typically within the acceptable dosage range. Compounds of the
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instant invention may alternatively be used sequentially with known
pharmaceutically acceptable agent(s) when a combination formulation i.s
inappropriate.
The daily dosage will normally be determined by the
5 prescribing physician, who may vary the dosage according to the age,
weight, and response of the individual patient, a.s well as the severity of
the patient's condition.
In one exemplary application, a suitable amount of
compound is administered to a m~mm~l undergoing treatment for
10 cancer. Adrnini.stration 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/l~g of body weight to about 40 mgtkg of body weight
per day.
The compounds of the instant invention are also useful
15 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
20 terminus) and farnesyl pyrophosphate and, in one of the mixtures,
a compound of the instant invention. After the assay mixtures are
incubated for an sufficient period of time, well known in the art,
to allow the FPTase to farnesylate the substrate, the chemical content
of the assay mixture.s may be determined by well known immuno-
25 logical, radiochemical orchromatographic techniques. Becausethe 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 sub~strate in the a,ssay
30 containing the instant compound i.s 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 u~seful in identifying
tissue samples which contain farnesyl-protein transferase and quanti-
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tating the enzyme. Thus, potent inhibitor compounds of the instant
invention may be u~sed in an active site titration assay to determine the
quantity of enzyme in the sample. A serie~s of .samples composed of
aliquots of a tissue extract containing an unknown amount of farne,syl-
5 protein transferase, an excess amount of a known substrate of FPTa~se
(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
10 that has a Ki Isubstantially smaller than the concentration of enzyme in
the assay vessel) required to inhibit the enzymatic activity of the sample
by 50% is approximately equal to half of the concentration of the
enzyme in that particular sample.
EXAMPLE 1
(+)-1 -(4-CYANOBENZYL)-S-1 ( I -HYDROXY-3-PHENYL)-2-
PROPYNYLlIMIDAZOLE HYDROCHLORIDE
20 Step A: Preparation of l-triphenylmethyl-4-
(hydroxymethyl)imidazole
To a solution of 4-(hydroxymethyl)imidazole
hydrochloride (35.0 g, 260 mmol) in 250 mL of dry DMF at room
temperature was added triethylamine (90.6 mL, 650 mmol). A white
25 solid precipitated from the solution. Chlorotriphenylmethane (76.1 g,
273 mmol) in 500 mL of DMF was added dropwise. The reaction
mixture was .stirred for 20 hour.s, poured over ice, filtered, and washed
with ice water. The resulting product wa,s slurried with cold dioxane,
filtered, and dried in vacuo to provide the titled product as a white solid
30 which was sufficiently pure for use in the next ,step.
Step B: Preparation of l-triphenylmethyl-4-
(acetoxymethyl)imidazole
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Alcohol from Step A (260 mmol, prepared above) was
suspended in 500 mL of pyridine. Acetic anhydride (74 mL, 7~0
mmol) wa.s added dropwi.se, and the reaction was stirred for 4~ hours
during which it became homogeneous. The solution was poured into 2
S L of EtOAc, washed with water (3 x 1 L), 5% aq. HCI 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 was
isolated a~s a white powder (85.X g, 86% yield for two ~teps) which wa~s
sufficiently pure for use in the next reaction.
Step C: Preparation of 1-(4-cyanobenzyl)-S-
(acetoxymethyl)imidazole hydrobromide
A solution of the product from Step B (85.8 g, 225 mmol)
and ~-bromo-p-tolunitrile (50.1 g, 232 mmol) in 500 mL of EtOAc was
lS stirred at 60 ~C for 20 hours, during which a pale yellow precipitate
formed. The reaction wa.s cooled to room temperature and filtered to
provide the solid imidazolium bromide salt. The filtrate was concen-
trated in vacuo to a volume 200 mL, reheated at 60~C for two hours,
cooled to room temperature, and filtered again. The filtrate was
20 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 V~lCUo to provide
25 a white solid which was triturated with hexane to remove soluble
materials. Removal of residual solvents in vacuo provided the titled
product hydrobromide a~s a white solid (50.4 g, 67% yield, ~9% purity
by HPLC) which was used in the next step without further purification.
30 Step D: Preparation of 1-(4-cyanobenzyl)-S-
(hydroxymethyl)imidazole
- To a solution of the acetate from Step C (50.4 g, 150
mmol) in l .S L of 3: I THF/water at 0 ~C was added lithium hydroxide
monohydrate (18.9 g, 450 mmol). After one hour, the reaction was
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concentrated in vacuo, diluted with EtOAc (3 L), and washed with
water, .sat. a~. NaHCO~ and brine. The solution was then dried
(Na2SO4), filtered, and concentrated in vacuo to provide the crude
product (26.2 g, ~2% yield) as a pale yellow fluffy solid which was
~S sufficiently pure for use in the next ~itep without further purification.
Step E: Preparation of 1-(4-cyanobenzyl)-~S-
imidazolecarboxaldehyde
To a solution of the alcohol from Step D (21.5 g,
101 mmol) in 500 mL of DMSO at room temperature was added
triethylamine (56 mL, 402 mmol), then SO3-pyridine complex (40.5
g, 254 mmol). After 45 minutes, the reaction was poured into 2.5 L
of EtOAc, washed with water (4 x 1 L) and brine, dried (Na2SO4),
filtered, and concentrated in vacuo to provide the aldehyde (1~.7 g,
1~ ~8% yield) as a white powder which was sufficiently pure for use in
the next step without further purification.
Step F: Preparation of (+)-1-(4-cyanobenzyl)-5-[(1-hydroxy-3-
phenyl)-2-propynyllimidazole hydrochloride
To a solution of phenylacetylene (0.172 mL, 1.56 mmol)
in 5 mL of THF at 0 ~C was added n-butyllithium (0.530 mL, 2.5 M in
hexanes, 1.32 mmol). After 15 minutes, the aldehyde from Step E (254
mg, 1.20 mmol) was added, and the reaction was stirred for 30 minutes.
The reaction was quenched with sat. aq. NaHCO3, poured into EtOAc,
washed with sat. aq. NaHCO3 and brine, dried (Na2SO4), filtered, and
concentrated in vacuo. The resulting product was purified by silica gel
chromatography (35-50% acetone/CH2CI2) to provide 1 ~7 mg of the
desired alcohol. A portion of this was taken up in CH2CI2 and treated
with excess 1 M HCI/ether solution, and concentrated in vacuo. The
titled product hydrochloride wa.s isolated as a white ~olid.
- FAB mas.~i spectrum m/e 314 (M+l).
Analysis calculated for C2~HI~N3O ~ 1.0 HCI - 0.90 H2O:
C, 65.63; H, 4.90; N, 11.4~;
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Found: C, 65.81; H, 4.98; N, 1 1.17.
EXAMPLE 2
(+)-1 -(4-CYANOBENZYL)-5-~( 1 -HYDROXY-4-PHENYL)-3-
BUTYNYLlIMIDAZOLE HYDROCHLORIDE
To a solution of t-butyllithium in 1.5 mL of THF (0.78 mL
of 1.7 M in pentane, 1.32 mmol) at -78~C was added tetramethylethyl-
enediamine (0.199 mL, 1.32 mmol) and 1-phenyl-1-propyne (0.150
mL, 1.20 mmol). The solution was warmed to 0 ~C for one hour, then
cooled to -78 ~C. The aldehyde from Step E of Example 1 (225 mg,
1.07 mmol) on 1.0 mL THF was added, and the reaction allowed to
warm to 0 ~C. After 30 minutes, the reaction was quenched with sat.
aq. NaHCO3, poured into EtOAc, washed with sat. aq. NaHCO3 and
brine, dried (Na2SO4), filtered, and concentrated in vacuo. The product
was purified by silica gel chromatography (3-4% MeOH/CH2CI2), then
taken up in CH2CI2 and treated with excess 1 M HCI/ether solution, and
concentrated in vacuo to provide the titled product hydrochloride (48
mg) as a pale yellow foam.
FAB mass spectrum m/e 32~ (M+l).
Analysis calculated for C2lH17N3O ~ 1.10 HCl ~ 0.10 Et2O:
C, 68.56; H, 5.14; N, 11.21;
Found: C, 68.30; H, 5.04; N, 11.06.
EXAMPLE 3
(+)-3-(4-CYANOBENZYL)-4-l ( 1 -HYDROXY-3-PHENYL)-2-
PROPYNYLlPYRIDINE HYDROCHLORIDE
- Step A: Preparation of 3-(4-cyanobenzyl)pyridin-4-carboxylic
acid methyl ester
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A solution of 4-cyanobenzyl bromide (625 mg, 3.27 mmol)
in dry THF (4mL) was added slowly over~3 min. to a suspension of
activated Zn (dust; 250 mg) in dry THF (2 mL) at O~ under an argon
atmosphere. The ice-bath was removed and the slurry was stirred at
S room temperature for a further 30 min. Then 3-bromopyridin-4-
carboxylic acid methyl ester (~40 mg. 2.5 mmol) followed by
dichlorobis(triphenylphosphine)nickel (Il) (50 mg). The resultant
reddish-brown mixture was stirred for 3h at ~40-45~C. The mixture
was cooled and distributed between EtOAc (l00 ml) and 5% aqueou~
citric acid (50 mL). The organic layer was washed with H2O (2X50
mL), dried with Na2SO4. After evaporation of the solvent the residue
was purified on silica gel, eluting with 35% EtOAc in hexane to give
420 mg as a clear gum. FAB ms (M+l) 253.
Step B: Preparation of 3-(4-cyanobenzyl)-4-
(hydroxymethyl)pyridine
The title compound was obtained by sodium borohydride
(300 mg) reduction of the ester from Step A (415 mg) in methanol
(5 mL) at room temperature. After stirring for 4 h the solution was
evaporated and the product was purified on silica gel, eluting with 2%
methanol in chloroform to give the title compound. FAB ms (M+l )
225.
Step C: Preparation of 3-(4-cyanobenzyl)-4-pyridinal
The title compound was obtained by activated manganese
dioxide (1.0g) oxidation of the alcohol from Step B (240 mg, 1.07
mmol) in dioxane (10 mL) at reflux for 30 min. Filtration and
evaporation of the solvent provided title compound, mp ~0-~s3OC.
Step D: Preparation of (+)-3-(4-cyanobenzyl)-4-[(1-hydroxy-3-
phenyl)-2-propynyllpyridine hydrochloride
The titled compound is prepared from the pyridinal from
Step C using the procedured in Step F of Example 1. The product is
purified by silica gel chromatography, then taken up in CH2CI2 and
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treated with excess 1 M HCl/ether solution, and concentrated i~ cuo to
provide the titled product hydrochloride.
EXAMPLE 4
1 -(4-BIPHENYLMETHYL)-5-(4-CYANOBENZYL)IMIDAZOLE
HYDROCHLORIDE SALT
Step A: 1 -Trityl-4-(4-Cyanobenzyl)-imidazole.
To a suspension of activated zinc dust (3.57g, 54.98mmol)
in THF (50ml)was added dibromoethane (0.315m1, 3.60mmol) and the
reaction stirred under argon at 20~C. The suspension was cooled to 0~C
and (x bromo-p-toluinitrile (9.33g, 47.6mmol) in THF (lOOml) was
added dropwise over a period of 10 min. The reaction was then allowed
to stir at 20~C for 6hr and bi,s(triphenylphosphine)Nickel II chloride
(2.4g, 3.64mmol) and 5 -iodotrityl imidazole (15.95g, 36.6mmol) was
added in one portion.The resulting mixture was stirred 16hr at 20~C
and then quenched by addition of saturated NH4CI solution (lOOml) and
the mixture stirred for 2 hours. Saturated NaHCO3 solution was added
to give a pH of P~ and the solution wa,s extracted with EtOAc (2x250ml),
dried MgSO4 and the solvent evaporated in vacuo. The residue was
chromatographed (sio2~ 0-20% EtOAc/CH2Cl2 to afford the title
compound as a white solid.
1H NMR ~ CDC13 (7.54 (2H, d, J=7.9Hz), 7.38(1H, s), 7.36-7.29 (1 lH,
m), 7.15-7.09(6H, m), 6.58(1H, s), and 3.93(2H, s)ppm.
Step B: 1 -(4-Biphenylmethyl)-5-(4-cyanobenzyl)imidazole
hydochloride salt
To 1-Trityl-4-(4-Cyanobenzyl)-imidazole (60~.~ mg,
1.43 mmol) in acetonitrile (2 ml) was added 4-chloromethyl biphenyl
(290mg, 1.43 mmol) and the mixture heated at 55~C for 16 hours. The
- residue was dissolved in methanol (30 ml) and heated at reflux for 20
mins, cooled and evaporated to dryness. The residue was partitioned
between saturated NaHCO3 solution and CH2C12. The organic layer
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was dried (MgSO4) and the solvent evaporated in vacuo. The re~idue
wa~ chromatographed (SiO2, 5% methanol in CH2C12) to afford the
imidazole which was converted to the HCI salt by treatment with one
equivalent of HCI in a4ueous acetonitrile. Evaporation of Isolvent in
5 vacuo afforded the title compound as a white powder.
Anal. Calcd for C24HlgN3- 1.00 HCI:
C, 74.70; H, 5.22; N, 10.~9.
Found: C, 74.70; H, 5.31; N, 10.77.
FAB MS 350 (MH+)
10 lH NMR CD30D ~ 9.03(1H, s), 7.65-7.50(5H, m), 7.44(2H, t,
J=7.5Hz), 7.39(1H, s), 7.35(1H, t, J=7.3Hz), 7.26(2H, d, J=~.lHz),
7.20(2H, d, J=X.lHz), 5.42(2H, s), and 4.17(2H, ,s) ppm.
EXAMPLE 5
I l -(4-CYANOBENZYL)IMIDAZOL-5-YLl(~ 1.1 '-BIPHENYLl-4-
YL)METHANOL
A Grignard reagent, freshly prepared from 4-bromo[l,l'-
20 biphenyl] (116 mg, 500 ,umol) and magnesium turnings (1~ mg, 730
,umol) in dry THF (500 ,ul) was added to a dry Argon-purged 3mL flask
containing the aldehyde (105 mg, 500 }lmol) in dry THF (200 ~L) with
vigorous stirring at room temperature. After I hour the reaction was
quenched with sat. NH4CI (5 mL) and distributed between EtOAc (50
25 mL) and H2O (50 mL). The organic phase was evaporated and the
residue was chromatographed on silica gel (CHCI3-MeOH (20:1)) to
yield title (117 mg).
FAB ms (M+l) 366.25.
Anal. Calc. for C24H IgN3O-0.10 CHCI3-0.10 CH3OH;
C, 76.37: H, 5.16: N, 11.04.
Found: C, 76.13; H, 5:10; N, 10.76.
EXAMPLE 6
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11-(4-CYANOBENZYL)IMIDAZOL-5-YLl(l l.l'-BIPHENYLl-4-
YL)KETONE
The alcohol (Example 5) (105 mg, 22~s ,umol) wa,s added to
5 dioxane (3 mL) and activated MnO2 (300 mg) and the black mixture
wa~s stirred at reflux for 2 hr. The mixture was filtered and the clear
filtrate was evaporated and the residue was chromatographed on silica
gel (CHCl3-MeOH (30:1)) to yield title (35 mg).
FAB ms (M+l) 364.07.
10 Anal. Calc. for C24HI7N3O-0.35 CHCI3;
C, 72.17; H, 4.32; N, 10.37.
Found: C, 71.87; H, 4.45; N, 10.29.
EXAMPLE 7
1 - ~ (4-CYANOBENZYL)- 1 H-IMIDAZOL-5-Yl:,lETHYL ~ -4-
PHENYL IMIDAZOLE BIS HYDROCHLORIDE SALT
N
NC~~
Step A: lH-Imidazole-4- acetic acid methyl ester hydrochloride
A solution of lH-imidazole-4-acetic acid hydrochloride
(4.00g, 24.6 mmol) in methanol (100 ml) wa.s saturated with ga,seous
hydrogen chloride. The resulting solution was allowed to stand at room
temperature for 1~ hrs. The solvent was evaporated in vacuo to afford
the title compound as a white solid.
lH NMR CDC13, ~ 5(lH, ,s), 7.45(1H, s), 3.~9(2H, s) and 3.75(3H,
s) ppm.
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Step B: l-(Triphenylmethyl)-lH-imidazol-4-ylacetic acid methyl
ester
To a solution of the product from Step A (24.85g,
0.141mol) in DMF (l lSml) was added triethylamine (57.2 ml,
5 0.412mol) and triphenylmethyl bromide (55.3g, 0.171mol) and the
suspension was stirred for 24 hrs. After this time, the reaction mixture
was diluted with EtOAc and water. The organic phase was washed with
saturated aqueous NaHCO3, dried (Na2SO4) and the solvent evaporated
in vacuo. The residue was purified by chromatography (sio2~ gradient
10 elution, 0-100% EtOAc in hexanes; ) to provide the title compound as a
white solid.
IH NMR CDC13, ~ 7.35(1H, s)~ 7.31(9H, m), 7.22(6H, m), 6.76(1H,
s), 3.68(3H, s) and 3.60(2H, s) ppm.
~5 Step C: [1 -(4-Cyanobenzyl)- 1 H-imidazol-5-yl]acetic acid methyl
ester.
To a solution of the product from Step B (8.00g,
20.9mmol) in acetonitrile (70 ml) was added 4-cyanobenzyl bromide
(4.10g, 20.92 mmol) and heated at 55~C for 3 hr. After this time, the
20 reaction was cooled to room temperature and the resulting imidazolium
salt was collected by filtration. The filtrate was heated at 55~C for
18hrs. The reaction mixture was cooled to room temperature and
evaporated in vacuo. To the residue was added EtOAc (70 ml) and
the resulting precipitate collected by filtration. The precipitated
25 imidazolium salt~s were combined, suspended in methanol (100 ml)
and heated to reflux for 30 min. After thi.s time, the solvent wa~s
removed in vac~o,. The resulting residue was suspended in EtOAc
(75ml) and the solid isolated by filtration and washed with EtOAc.
The solid was treated with saturated aqueous NaHCO3 solution
30 (300ml) and CH2C12 (300ml) and stirred at room temperature for
2 hrs. The organic layer was separated, dried (MgSO4) and
evaporated in vacuo to afford the title compound as a white solid
lHNMR CDC13, ~ 7.65(1H, d, J=8Hz), 7.53(1H, s), 7.15(1H, d.
J=8Hz), 7.04(1H, s), 5.24(2H, s), 3.62(3H, s) and 3.45(2H, s) ppm.
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-4X-
Step D: 5-11-(4-cyanobenzyl)-lH-imidazolyllethanol.
To a stirred solution of the ester from example step C,
(l.SOg, 5.8~mrnol), in methanol (20 ml) at 0~C, was added sodium
5 borohydride (l.OOg, 26.3mmol) portionwise over 5 min. The reaction
was stirred at 0~C for 1 hr and then at room temperature for an
additional 1 hr. The reaction was quenched by the addition of saturated
NH4CI solution and the methanol evaporated in vacuo.. The residue was
partitioned between EtOAc and saturated NaHC03 solution and the
10 organic extracts dried, (MgS04) and evaporated in vacuo. The residue
was purified by chromatography (sio27 gradient elution, 4 to 10%
methanol in methylene chloride) to afford the title compound as a white
solid.
1 H NMR CDC13 ~ 7.64(2H, d, J=8.2Hz), 7.57(1 H, s), 7.11 (2H, d,
15 J=8.2Hz), 6.97(1H, s), 5.23(2H, s), 3.79(2H, t, J=6.2Hz), 2.66(2H, t,
J=6.2Hz) ppm.
Step E: 5~ (4-Cyanobenzyl~-imidazolyl)ethylmethanesulfonate.
A solution of 5-[1-(4-cyanobenzyl)-lH-imidazolyl]ethanol
20 (0.500 g, 2.20 mmol) in methylene chloride (6 ml) at 0~C was treated
with Hunig's base (0.460ml, 2.64mmol) and methanesulfonyl chloride
(0.204ml, 2.64mmol). After 2 hrs, the reaction was quenched by
addition of saturated NaHC03 solution (SOml) and the mixture
extracted with methylene chloride (50ml), dried (MgS04) and the
25 solvent evaporated in vacuo. The title compound was used without
furthur purification.
lH NMR CDC13 ~ 7.69 (lH, s) 7.66(2H, d, J=8.2Hz), 7.15 (2H, d,
J=8.2Hz), 7.04(1H, s), 5.24(2H, s), 4.31(2H, t, J=6.7Hz), 2.96(3H, s),
and 2.88(2H, t, J=6.6Hz)ppm.
Step F~ [1 -(4-Cyanobenzyl)- 1 H-imidazol-5 -yl]ethyl ) -4-phenyl - imidazole bis hydrochloride salt.
To a suspension of sodium hydride (14.2mg, 60%
dispersion in mineral oil, 0.356mmol) in DMF (0.30 ml) at 0~C was
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added 4-phenyl imidazole (4~.~mg, 0.339mmol), and stirred for 20
mins. A solution of the mesylate from step E (lOOmg, 0.339mmol) in
DMF (0.50ml) was added to the Isolution and stirring continued at 0~C
for 1 hr and then at room temperature for 16 hrs. The reaction was
5 quenched with saturated ammonium chloride solution (O.lOml). and
the the solvent evaporated in vacuo. The re~sidue was purified by
chromatography (sio2~ gradient elution, 2-5% ammonium hydoxide:
acetonitrile. The resulting material was converted to the HCI salt by
treating an EtOAc solution of the imidazole with gasseouls HCI and
10 evaporating the ~solvent in vacuo.
.. . . . . . .. . . .. . .. .
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Anal. Calcd for C22H 19N5-2-00HCl- 1 -50H20:
C, Sg.29; H, 5.34; N, 15.45.
Found: C, 58.24; H, 5.47; N, 15.4~.
FAB HRMS exact mass calcd for C22H20N5 354.171 g71 (MH+); found
5 354.171948.
lH NMR CD30D ~ ~.93 (lH, s), 8.75(1H, s), 7.~6(1H, s), 7.76(2H, d,
J=7.9Hz), 7.69(2H, d, 7.1Hz), 7.65-7.35(6H, m), 5.61(2H, s) and
4.53(2H,m)ppm.
10 In vitro inhibition of ras farne,syl transferase
Assays offarnesyl-protein transferase.
Partially purified bovine FPTase and Ras peptides
(Ras-CVLS, Ras-CVIM and Ras-CAIL) were prepared as described
by Schaber et ah, J. Biol. Chem. 265:14701-14704 (1990), Pompliano,
15 et al., Biochemistry 31:3800 (1992) and Gibbs et al., PNAS U.S.A.
~6:6630-6634 (1989), respectively. Bovine FPTa.se was assayed in a
volume of 100 ~1 containing 100 mM N-(2-hydroxy ethyl) piperazine-
N'-(2-ethane sulfonic acid) (HEPl~S), pH 7.4, 5 mM MgC12, 5 mM
dithiothreitol (DTT), 100 mM [3H]-farnesyl diphosphate ([3H]-FPP; 740
20 CBq/mmol, New England Nuclear), 650 nM Ras-CVLS and 10 ~g/ml
FPTase at 31 ~C for 60 min. 3~eactions 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
25 was linear with respect to both substrates, FPTase levels and time; less
than 10% of the [3H]-FPP was utilized during the reaction period.
Purified compounds were dissolved in 100% dimethyl sulfoxide
(DMSO) and were diluted 20-fold into the assay. Percentage inhibition
is measured by the amount of incorporation of radioactivity in the
30 presence of the test compound when compared to the amount of
incorporation in the absence of the test compound.
- Human FPTa~se was prepared as described by Omer et al.,
Biochemistry 32:5167-5176 (1993). Human FPTase activity was
assayed as described above with the exception that 0.1 % (w/v)
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-5 1 -
polyethylene glycol 20,000, 10 ~lM ZnC12 and 100 nM Ras-CVIM were
added to the reaction mixture. Reactions were performed for 30 min.,
stopped with 100 ~1 of 30% (v/v) trichloroacetic acid (TCA) in ethanol
and processed as described above for the bovine enzyme.
S The compounds of the instant invention described in the
above Examples 1-7 were tested for inhibitory activity against human
FPTase by the assay described above and were found to have IC50 of
50 ~lM.
In vivo ras farnesylation assay
The cell line u~ed 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 ah,
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-free DMEM supple-
meted with 10% regular DMEM, 2~o fetal bovine serum and 400
mCi[35S]methionine (1000 Ci/mmol). After an additional 20 hours, the
cells are lysed in 1 ml Iysis buffer (1% 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 Iysates cleared by centrifugation at
100,000 x g for 45 min. Aliquots of Iysates containing equal numbers
of acid-precipitable count.s are bought to 1 ml with IP buffer (lysis
buffer lacking DTT) and immunoprecipitated with the ra~s-specific
monoclonal antibody Y13-259 (Furth, M.E. et ah, J. Virol. 43:294-304,
(19X2)). Following a 2 hour antibody incubation at 4~C. 200 ml of a
25% suspension of protein A-Sepharose coated with rabbit anti rat IgG
is added for 45 min. The immunoprecipitates are washed four times
with IP buffer (20 nM HEPES, pH 7.5/1 mM EDTA/l % 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
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farne~ylated and nonfarnesylated ras proteins are compared to
determine the percent inhibition of farnesyl transfer to protein.
In viv(~ ~rowth inhibition assay
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-7~af, or v-mos oncogene is tested. Cells transformed by v-Raf
and v-Mos maybe included in the analysis to evaluate the specificity of
instant compounds for Ras-induced cell transformation.
Rat 1 cells transformed with either v-ras, v-raf, or v-mos
are seeded at a density of 1 x 104 cells per plate (35 mm in diameter) in
a 0.3% top agarose layer in medium A (Dulbecco's modified Eagle's
medium supplemented with 10% fetal bovine serum) over a bottom
agarose layer (0.6%). Both layers contain 0.1% methanol or an
appropriate concentration of the instant compound (dissolved in
methanol at 1000 times the final concentration used in the assay).
The cells are fed twice weekly with 0.5 ml of medium A containing
0.1% methanol or the concentration of the instant compound.
Photomicrographs are taken 16 days after the cultures are seeded
and comparisons are made.
