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
BACKGROUND OF THE ~VENTION
The present invention relates to compound.s which
inhibit farnesyl protein transferase, a protein which is implicated in
the oncogenic pathway mediated by Ra.s. The Ra.s protein,s (Ha-Ras,
Ki4a-Ras, Ki4b-Ras and N-Ras) are part of a signalling pathway that
links cell surface growth factor receptors to nuclear signals initiating
cellular proliferation. Biological and biochemical .studie.~i of Ras action
indicate that Ra.s functions like a G-regulatory protein. In the inactive
~state, Ras i.s bound to GDP. Upon growth factor receptor activation R~
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 ~-ignal is terminated by the intrinsic
GTPase activity of Ras, which returns the protein to its inactive GDP
bound form (D.R. Lowy and D.M. Willumsen, AM~1. Rel!. BiOChenl.
~2:~51-~91 (1993)). Mutated ra~ genes (Ha-7a~, Ki4a-ru~, Ki4b-ras
and N-ras) are found in many human cancers, including colorectal
carcinoma, exocrine pancreatic carcinoma, and myeloid leukemia.s.
The protein products of the~e genes are defective in their GTPase
activity and con.stitutively transmit a growth .stimulatory signal.
Ras must be localized to the plasma membrane for
both normal and oncogenic functions. At least 3 post-translational
modifications are involved with Ras membrane localization, and all
3 modifications occur at the C-te~ninu,s of Ras. The Ra~ C-terminus
contain~ a sequence motif termed a "CAA~" or "Cys-Aaal-Aaa2-Xaa"
box (Cy.s i.s cy.steine, Aaa i~i an aliphatic amino acid, the Xaa i~ any
amino acid) (Willumsen et al., Nature 310:5~3-5~6 ( 19R4)). Depend-
ing on the .specific .sequence? this motif serves as ~ ~;ignal ~sequence for
the enzymes farnesyl-protein tran,sferase or geranylgeranyl-protein
transferase, which catalyze the alkylation of the cy,steine residue of the
CAAX motif with a Cls or C2() isoprenoid, re.spectively. (S. Clarke.~
AMM. Rel~. B~ he~7?. fjl :355-3~6 (1992); W.R. Schafer ~nd J. Rine, Ann.
CA 022~0143 1998-09-2~
WO g7/36583 PCT/US97/05170
Rev. Genetics 30:209-237 (1992)). Ra.s proteins are known to undergo
post-translational farnesylation. Other farnesylated proteins include the
Ras-related GTP-binding proteins ~such as Rho, fungal mating factors,
the nuclear lamins, and the gamma subunit of transducin. James, et al.,
5 J. Bivl. Chem. 269, 14182 (1994) have identified a peroxilsome
associated protein Pxf which i~ also ~arnesyl-
ated. James. et al., have also .~uggested that there are farnesylated
proteins of unknown structure and function in addition to those listed
above.
Inhibition of farnesyl-protein transfera~e has been
shown to block the growth of ~as-transformed cells in soft ag~r and to
modify other aspects of their transformed phenotype. It has also been
demonstrated that certain inhibitors of farne!iyl-protein transfera.se
selectively block the processing of the Ras oncoprotein intracellularly
15 (N.E. Kohl et al., Science, 260:1934-1937 (1993) and G.L. Jame.s et al.,
Science, 260:1937-1942 (1993). Recently, it has been ~hown that an
inhibitor of farnesyl-protein transferase blocks the growth of ras-
dependent tumors in nude mice (N.E. Kohl et al., P~ oc. Natl. Acad. S~i
U.S.A., 91:9141-9145 (1994) and induces regression of m~mm~ry and
20 salivary carcinomas in )~as transgenic mice (N.E. Kohl et al., Natu) e
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., ihid; Casey et al., ihid; Schafer
25 et al., Science 245:379 (1989)). These drugs inhibit HMG-CoA
reductase, the rate limiting enzyme for the production of poly-
isoprenoids including farnesyl pyrophosphate. Farnesyl-protein
transfera.se utilize~s farnesyl pyrophosphate to covalently modify
the Cys thiol group of the Ras CAAX box with a farne.syl group (Reiss
30 et al., Cell, 62~ (1990); Schaber et al., J. Biol. Chem.. 265:14701-
14704 (1990); Schafer et al., Science, 249:1133-1139 (1990); Manne
et al., P~oc. Natl. Acad. Sci USA, ~7:7541-7545 (1990)). Inhibition of
farnesyl pyrophosphate biosynthesis by inhibitin~ HMG-CoA reductase
blocks Ras membrane localization in cultured cells. However, direct
CA 02250l43 l998-09-25
WO 97/36583 PCT/US97/05170
inhibition of farnesyl-protein transfera~se would be more ~specific, and
thu,s preferable.
Inhibitors of farne~syl-protein transferase (FPTase) have
been described in two general classe~s. The fir~st are analogs of farnesyl
diphosphate (FPP), while the second cla,ss of inhibitors is related to the
protein substrates (e.g., Ras) for the enzyme. The peptide derived
inhibitor.s that have been de~scribed are generally cysteine containing
molecule~s that are related to the CAAX motif that is the signal for
protein prenylation. (Schaber et al., ibid; Reiss et. c~l., ihid; Reiss
et al., PNAS, 88:732-736 (1991)). Such inhibitors may inhibit protein
prenylation while serving as alternate substrates for the famesyl-protei
tran~sfera~se enzyme, or may be purely competitive inhibitor~s (U.S.
Patent 5,141,~51, University of Texa~s; 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 inhibitors
also inhibit the proliferation of vascular ,smooth muscle cells and are
therefore useful in the prevention and treatment of arteriosclero~sis
and diabetic disturbance of blood vessel,s (JP H7-112930).
It ha,s recently been disclosed that certain tricyclic
compound,s which optionally incorporate a piperidine moiety
are inhibitors of FPTase (WO 95/10514, WO 95/10515 and
WO 95/10516). Imidazole-containing inhibitors of farne.syl
protein transferase have al~so been disclo~sed (WO 95/09001
and EP 0 675 112 Al).
SUMMARY OF THE INVENTION
The present invention addresse~s a compound of formula 1:
- V Al (CR1 a2) A2(C R 1b2)n -(W)- (CR 2~A - (CR 2)p
R4
I
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or a pharmaceutically acceptable salt thereof, wherein:
Rla, R1b and R2 are independently selected from
the group consisting of: hydrogen, aryl, heterocyclyl, C3-Clo
S cycloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, R8O-, R9S(o)m-
~(R~)2NC(O)-, R~C(O)NR8-, CN, NO2, (R~)2NC(NRg)-, R8C(O)-,
R~OC(O)-, N3, -N(R~)2, R9OC(o)NR8- and Cl-C6 alkyl, unsubstituted
or substituted by 1-3 groups selected from the group con.sisting of:
halo. aryl, heterocyclyl, C3-Clo cycloalkyl, C2-c6 alkenyl, C2-C6
10 alkynyl, R8O-, R9S(o)m-~ R~C(O)NR~-, CN, (R~)2NC(NR~)-,
R~C(O)-, R8OC(O)-, N3, -N(R8)2 and R9OC(o)NR~-;
R3 and R4 are independently selected from the group
consisting of: H, F, Cl, Br, -NR82, CF3, NO2, R8O-, R9S(o)m-
~
15 (R~)2NC(O)-, R8C(O)NH-, H2NC(NH)-, R~C(O)-, R~sOC(O)-, N3, CN,
R9OC(o)NR8-, Cl-C20 alkyl, substituted or unsubstituted aryl
and substituted or unsubstituted heterocyclyl;
A3 is selected from: ~NRSs(o)m- or -S(O)mNRS-
~
20 with m equal to 0, 1 or 2, and RS selected from the group consisting of:hydrogen, unsubstituted or substituted aryl, unsubstituted or substituted
heterocyclyl, un~substituted or substituted C3-Clo cycloalkyl, ~md Cl-C6
alkyl, unsubstituted or substituted with 1-3 members selected from the
group consisting of: unsubstituted or substituted aryl, unsubstituted or
25 sub.stituted heterocyclyl, unsubstituted or substituted C3-Clo cycloalkyl,
-N(R~)2, -CF3, -NO2, (R~)O-, (R9)S(o)m-~ (R~)C(O)NH-, H2NC(NH)-
, (R~)C(O)-, (R~)OC(O)-, N3, CN and (R9)OC(O)NR~-;
R6 and R7 are independently selected from the group
30 consisting of: hydrogen, ~ryl. heterocyclyl, C3-CIo cycloalkyl,
C2-C6 alkenyl, C2-C6 alkynyl, C1 6 perfluoroalkyl, F, Cl, Br,
R~O-, R9S(o)m-, R~sC(O)NRPi-, CN, N02, (RX)2NC(NR~)-,
R~C(O)-, R~OC(O)-, N3, -N(R~s)2, R90C(o)NR~- and Cl-C6
alkyl unsubstituted or substituted by 1-3 groups selected from:
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aryl, heterocyclyl, C3-Clo cycloalkyl, C2-C6 alkenyl, C2-C6
alkynyl, perfluoroalkyl, F, Cl, Br, R~O-, R9S(o)m-~ RXC(O)NRx-,
CN, (RP~)2NC(NR~)-, R~C(O)-, RXOC(O)-, N3, -N(RX)2 and
R9OC(o)NR~¢-;
s
each R~s ils independently selected from hydrogen,
Cl-C6 alkyl, aryl and aralkyl;
each R9 is independently selected from Cl-C6 alkyl and
10 aryl;
A 1 and A2 are independently ,selected from the group
con,sisting of: a bond, -CH=CH-, -C~C-, -C(O)-, -C(O)NR~-,
-NR~C(O)-, -O-~ -N(R8)-, -S(O)2N(RX)-, -N(R~)s(o)2-~ and S(O)m;~5
X represents aryl or heteroaryl;
V is ,selected *om the group consisting of: hydrogen,
heterocyclyl, aryl, Cl-C2o alkyl wherein from 0 to 4 carbon atoms are
20 replaced with a heteroatom selected from O, S, and N~ and C2-C20
alkenyl, provided that V i,s not hydrogen if Al is S(O)m and V is not
hydrogen if ~1 is a bond, n is 0 and A2 i.s S(O)m;
W represents heterocyclyl;~5
each n and p independently represents 0, 1, 2, 3 or 4;
r is 0 to 5, provided that r is 0 when V is hydrogen, and
ti,s0Or 1.
~0 DETAILED DESCRIPTION OF THE INVENTION
The compounds of thi,s invention are u~seful in the inhibition
of farnesyl-protein tran.sfera.se and the farnesylation of the oncogene
protein Ras, and thu~ are useful for the treatment of cancer.
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The compounds of the present invention may have
asymmetric centers and occur as racemates, racemic mixtures and as
individual diastereomers, with all possible isomers, including optical
isomers, being included in the present invention.
When any variable (e.g. aryl, heterocycle, Rl, R2 etc.)
occurs more than one time in any constituent, 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 atom~i~ or 1-6 carbon atoms if unspecified. Cycloalkyl means
1-2 cabocyclic rings which are saturated and contain from 3-10 atoms.
"Halogen" or "halo" as used herein means fluoro, chloro,
bromo and iodo.
As u~sed 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 i~s 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 ~- to I 1-
membered bicyclic heterocyclic rings, either saturated or unsaturated,
aromatic, partially aromatic or non-aromatic, and which consist of
carbon atoms and from one to four heteroatom,s selected from the group
consisting of N, O, and S. Thus, it includes any bicyclic group in which
any of the above-defined heterocyclic ring~s i,s fused to a benzene ring.
The ring or ring system may be attached at any heteroatom or carbon
atom which results in a stable structure, and may contain 1-3 carbonyl
groups. Examples of ~such heterocycles include, but are not limited to,
azepinyl, benzimidazolyl, benzisoxazolyl, benzofurazanyl,
benzopyranyl~ benzothiopyranyl, benzofuryl, benzothiazolyl,
benzothienyl~ benzoxazolyl, chromanyl, cinnolinyl, dihydrobenzofuryl,
dihydrobenzothienyl, dihydrobenzothiopyranyl,
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dihydrobenzothiopyranyl sulfone, furyl, imidazolidinyl, imidazolinyl,
imidazolyl, indolinyl, indolyl, isochromanyl, isoindolinyl, isoquinolinyl,
isothiazolidinyl, isothiazolyl, isothiazolidinyl, morpholinyl,
naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, 2-oxopiperazinyl,
2-oxopiperdinyl, 2-oxopyrrolidinyl, piperidyl, piperazinyl, pyridyl,
pyrazinyl, pyrazolidinyl, pyrazolyl, pyridazinyl, pyrimidinyl,
pyrrolidinyl, pyrrolyl, ~uinazolinyl, quinolinyl, quinoxalinyl,
tetrahydrofuryl, tetrahydroisoquinolinyl, tetrahydroquinolinyl,
thiamorpholinyl, thiamorpholinyl sulfoxide, thiazolyl, thiazolinyl,
thienofuryl, thienothienyl, and thienyl.
"~Ieteroaryl" i~ a .sublset of heterocyclic, 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 heteroatoms selected from the group
consisting of N, O, and S. Example~i include benzimidazolyl,
benzisoxazolyl, benzofurazanyl, benzopyranyl, benzothiopyranyl,
benzofuryl, benzothiazolyl, benzothienyl, benzoxazolyl, chromanyl,
cinnolinyl, dihydrobenzofuryl, dihydrobenzothienyl,
dihydrobenzothiopyranyl, dihydrobenzothiopyranyl sulfone, furyl,
imidazolyl, indolinyl, indolyl, isochromanyl, isoindolinyl, iso~uinolinyl,
isothiazolyl, naphthyridinyl, oxadiazolyl, pyridyl, pyrazinyl, pyrazolyl.
pyridazinyl, pyrimidinyl, pyrrolyl, quinazolinyl, ~uinolinyl,
quinoxalinyl, tetrahydroiso4uinolinyl, tetrahydro4uinolinyl, thiazolyl,
thienofuryl, thienothienyl and thienyl.
Lines drawn into ring systems from substituents indicate
that the bond may be attached to any of the substitutable ring atoms.
The term "substituted" as used with respect to, e.g.,
substituted alkyl, substituted aryl, substituted heterocyclyl and
substituted cycloalkyl mean alkyl, aryl, heterocyclyl and cycloalkyl
group~, respectively. having from 1-3 substituents which are selected
from: halo, aryl, heterocyclyl, C3 1~) cycloalkyl, Cl ~ alkyl, C2 ~,
alkenyl, C2 ~, alkynyl, RXO-, R9S(o),n-, RXC(O)NRX-, CN,
(RX)~NC(NRx)-, RXC(O)-, RXOC(O)-, N~" -N(RX)2 and R~OC(O)NRX-.
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Preferably 1-2 groups are present on substituted alkyl,
substituted aryl, substituted heterocyclyl and sub.stituted cycloalkyl,
which are selected from: halo, aryl, RX0-, CN, R~C(0)- and -N(RX)2.
Preferably, R1a, Rlb and R2 are independently selected
S from: hydrogen, -N(R~)2, R~C(O)NR8- or unsubstituted or substituted
Cl-C6 alkyl wherein the substituent on the substituted Cl-C6 alkyl is
selected from unsubstituted or substituted aryl, -N(R~)2, R80- and
R~C(O)NR~- Preferably, R3 and R4 are selected from: hydrogen
and Cl-C6 alkyl.
Preferably, A3 represents NRSS(O),n, wherein m represents
2 and R5 represents hydrogen.
Preferably, R6 represents hydrogen, un~sub~stituted or
.substituted Cl-C6 alkyl.
Preferably R7 repre,sents H or un,substituted C~ ~ alkyl.
Preferably, R% represents H or C~ ~ alkyl, and R9 is C
alkyl.
Preferably, Al and A2 are independently selected from:
a bond, -C(O)NR8-, -NR~C(0)-, -0-, -N(RP~)-, -S(0)2N(R~)- and-
N(R~S(0)2--
Preferably X represents aryl and mo.st preferably phenyl.
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 is selected from imidazolyl and pyridyl.
Preferably, m is 2.
Preferably n and p are independently 0, 1, 2 or 3.
Preferably t is 1.
A ,sub,set of compounds of the invention is repre.sented by
formula Ia:
-
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~ (R6) R7
V - A1(CR1a2)nA2(CR1b2)~N~_~J R3
(CR 2)p--A - (CR 2)p{
whereln:
R3, R4, A3, RX, R9, m, n, p and r are as originally defined;
each Rla and R2 is independently ~elected from hydrogen
and C I -C6 alkyl;
each Rlb i,~i independently selected from: hydrogen, aryl,
10 heterocyclyl, C3 10 cycloalkyl, C2 ~ alkenyl, R~O-, -N(R~)2 and Cl-C6
alkyl unsubstituted or.substituted by aryl, heterocyclyl, cycloalkyl,
alkenyl, R8O- and -N(R8)2;
RS is selected from the ~roup consisting of: hydrogen and
15 Cl-C6 alkyl, unsubstituted or substituted with 1-3 member~i ~elected
from the group con.sisting of: unsubstituted or substituted aryl,
unsubstituted or substituted heterocyclyl. unsub~stituted Ol .~iubstituted
C3-Clo cycloalkyl, -N(R~)2, -CF3, -N02, (R~)O-, (R9)S(o),~,-,
(R8)C(O)NH-, H2NC(NH)-, (R~)C(O)-, (R~¢)OC(O)-, N3, CN and
20 (R9)OC(O)NR8-;
R6 i~s .~elected from: hydrogen, Cl-C6 alkyl, C2-C6
alkenyl, C2-C6 alkynyl, Cl-C6 perfluoroalkyl, ~, Cl, R~O-,
R~C(O)NRP~-, CN, N02, (R~)2N-C(NR~)-, R~sC(O)-, R~OC(O)-,
2~ -N(R8)2, or R9OC(o)NRg-, and Cl-C6 alkyl substituted by Cl-C6
perfluoroalkyl, R~O-, R~C(O)NR~-, (R~)2N-C(NR~)-, R~SC(O)-,
R~OC(O)-, -N(RX)2 and R9OC(o)NR~-;
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- I O-
R7 is hydrogen or unsubstituted Cl ~ alkyl;
A I and A2 are independently selected from: a bond,
-CH=CH-, -C_C-, -C(O)-, -C(O)NR8-, O, -N(R~)- and S(O)m;
and V is selected from: hydrogen; aryl; heterocyclyl
,selected from pyrrolidinyl, imidazolyl, pyridinyl, thiazolyl, pyridonyl,
2-oxopiperidinyl, indolyl, quinolinyl, i.soquinolinyl and thienyl; Cl-C20
alkyl wherein from 0 to 4 carbon atoms are replaced with a a hetero-
10 atom selected from O, S, and N, and C2-C20 alkenyl, provided that V is
not hydrogen if A1 is S(O)m and V is not hydrogen if AI i~i a bond and
A2 i,'; S(O)m.
A ~econd subset of compound.s of the present invention is
repre,sented by formula Ib:
V - A1 (CRl a2)nA2(CR 1b2)n ~W)~ (C R22)F NRs-s(o)m- (CR22h~CI~)
Ib R4
wherein:
R1a, Rlb, R2, Al, A2, R3, R4, R5, R6, R~, R~, m, n, p and r
20 are as originally defined;
R7 is selected from: hydrogen and unsubstituted Cl-C6
alkyl;
2~ V is selected from: hydrogen, heterocyclyl selected
from pyrrolidinyl, imidazolyl, pyridinyl, thiazolyl, pyridonyl,
2-oxopiperidinyl. indolyl, quinolinyl, isoquinolinyl, and thienyl, aryl,
Cl-C20 alkyl wherein from 0 to 4 carbon atom~i are replaced with a
heteroatom selected from O, S, and N, and C2-C~o alkenyl,
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provided that V is not hydrogen if A~ S(O)m and V i~ not hydrogen
if Al is a bond, n i~ 0 and A2 is S(O)m; and
S ~I reprelsents heterocyclyl ~elected from pyrrolidinyl,
pyridinyl, thiazolyl, pyridonyl, 2-oxopiperidinyl, indolyl, quinolinyl
and i~oquinolinyl.
A third embodiment of the invention i~s de~cribed in
10 accordance with formula Ic:
H
)~N
(C R 2)p -A - (C R 2)p~
whereln:
each R2 i~j independently selected from hydrogen and
Cl-C6 alkyl;
R3, R4, A3, RX, R9, m and p are as originally defined;
each R5 i,s selected from: hydrogen and Cl-C6 alkyl
unsubstituted or substituted with 1-3 group~ selected from unstituted
or sub~tituted aryl, unsubstituted or substituted heterocyclyl,
unsubstituted or ~ubstituted C3-clo cycloalkyl, -N(R~)2~ -CF3, -NO2.
(R~)O-, (R9)S(o)m-, (R~)C(O)NH-, H2NC(NH)-, (R~)C(O)-,
2~; (R~)OC(O)-, N~,~ -CN ~nd (R9)OC(O)NR~-;
and R6 i~ ~;elected from the group con~ ting of: hydrogen~
Cl-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, Cl-C6 perfluoroalkyl, F,
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- 1 2-
Cl, R8O-, R~C(O)NR8-, CN, NO2, (R~)2N-C(NRX)-~ R~C(O)-,
R80C(O)-, -N(RP~)2, or R9OC(o)NR8- and CI-C6 alkyl substituted by
Cl-C6 perfluoroalkyl, R8O-, R~C(O)NR8-, (R8)2N-C(NR8)-, R~C(O),
R8OC(O)-, -N(R~)2 or R9OC(o)NR8-.
A fourth subset of compounds of the invention is
represented by formula Id:
H
(CR22)p 7~3 - (CR22)p{
n4
NC n
wherein:
each R2 is independently selected from: hydrogen and Cl-C6 alkyl;
R3 and R4 are independently selected from H, F, Cl, Br,
N(RX)2, CF3, NO2, (R8)o-, (R9)S(o)m-~ (R8)C(O)NH-, H2N-C(NH)-,
1~ (R8)C(O)-, (R~)OC(O)-, N3, CN, (R9)OC(O)NR~-, Cl-C20 alkyl,
substituted or unsubstituted aryl, and substituted or unsubstituted
heterocyclyl;
A3 represents -NR5-S(o),- or-S(O)m-NR5-;
R5 is selected from: hydrogen and Cl-C6 alkyl,
unsub~tituted or substituted witha group selected from unsubstituted Ol
substituted aryl, unsubstituted or substituted heterocyclyl, unsubstituted
or substituted C3-Clo cycloalkyl, N(R~)2, CF3, NO2, (R~)O-,
2~ (R9)S(O)m-, (R~)C(O)NH-, H2N-C(NH)-, (R~)C(O)-, (R~)OC(O)-,
N3, CN (R9)OC(O)NR~¢-;
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and R~, R9, m and p are a!i originally defined.
Specific examples of compound.s of the invention are:
G--~N ~ N H SO2~
N C I
NC~ ~SO2NH~ "(~
N
NC ~ ~ NHSO
N
NC ~ ~ SO2NH
N
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-14-
Nl ~ ~~ ~CI ~N o~S--N~ I
NC NC
\~\N--'S~
NC Cl
NC ~N N(CH3)SO~
N~ Cl
NC ~3\ ~SO2N(CH3
N
NC ~3~ N(CH3)S~
;~ N~
NC ~'N ~SO2N(CH3)~
N
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~ O~ ~CI ~ of S-N~
NC NC
~N-S~
NC
and the pharmaceutically acceptable salt,s thereof.
S The pharmaceutically acceptable salts of the compounds of
this invention include the conventional non-toxic salts of the compounds
of this invention a~s forrned, e.g., from non-toxic inorganic or organic
acid~s. For example, such conventional non-toxic salts include those
derived from inorganic acids such a~i hydrochloric, hydrobromic,
~sulfuric, sulfamic, phosphoric, nitric and the like: ~nd the salt~i prepared
from organic acid.s such as acetic, propionic, ~uccinic, 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 .salt.s of the compound.s
of thi.s invention can be synthe~;ized from the compound.s of thi.s
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 ba~e with stoichiometric
amount.s or with ~n exce~,s of the desired .salt-forming inorganic or
organic acid in a suitable .solvent or variou.s combination~s of ~solvent~s.
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Reaction.s u.sed to generate the compounds of this
invention are prepared by employing reactions a,s shown in Schemes 1-
7, in addition to other standard manipulations such as ester hydrolysis,
cleavage of protecting groups, etc., as may be known in the literature
5 or exemplified in the experimental procedures. Substituents R and
R CH2-, as shown in the Scheme,s, repre,sent the sub,stituents R~, R9 and
other,s, depending on the compound of the instant invention that i,s being
~synthesized. The variable p' repre,sents p-l.
These reaction,s may be employed in a linear ,sec~uence to
10 provide the compounds of the invention or they may be used to
synthesize fragments which are ,subsequently joined by the alkylation
reactions described in the Schemes.
Synopsi,s of Scheme.s
The re~luisite intermediate.s are commercially available or
can be prepared according to literature procedures. The Schemes
illustrate the synthesi,s of certain preferred embodiments of the instant
invention, wherein the variable W is present a,s an imidazolyl moiety
that is sub,stituted. Substituted protected imidazole alkanols II can be
prepared by methods, such as those de,~cribed by F. Schneider, Z.
Physiol . Chem ., 3 :206-210 (1961) and C.P. Stewart, Biochem. Jou~ nal,
17:130-133(1923). Benzylation and deprotection of the imidazole
alkanol provides an intermediate which can be oxidized to the
corresponding aldehyde.
The aldehyde IV is reacted with a suitably substituted
amine. The interrnediate can then be reacted with a sulfinyl chloride.
Syntheses of suitably sub.stituted aldehydes are illustrated
wherein the variable W is present as a pyridyl moiety. Similar synthetic
strategies for preparing alkanols that incorporate other heterocyclic
moieties for variable W are also well known in the art.
The ~sulfinamide can be formed by converting a hydroxyl
group to a .sulfinyl chloride, and then reacting the sulfinyl chloride with
the appropriately ~substituted amine. The re.sulting .sulfinamide can
thereafter be oxidized with, e.g., periodate, to produce sulfonamides in
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accordance with fonnula I. Likewi~e, by reacting the precur.sor amine
with an alkylating agent, .~ubstitution on the sulfonamide nitrogen atom
can be realized.
SCHEME 1
(CR22)pl-cH2oH Prot1X Et3N Prot 'N~/~>
H DMF lla
(CR22)p,-CH20Ac R6~EtOAc
AC20, Py ,=1
Prot1~N~ N 2. N-deprotect
~CR 2)p-CH20Ac N~'~(CR 2)p-CH20H
N~ LiOH >
R6~ THF, H20 ~
111
~(CR 2)p-CHO (CR22)p-CH2NHCH3
SO3 Py, Et3N ,=~) CH3NH2 N
- DMSO ~ Na(AcO)3B~
IV V
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SCHEME 2
OH
DEAD ~ SC(O)CH3
[~PPh3 CH3C(O)SH
Ac20 /S(O)CI Et~N
CH2CI2 ¢~ CH2CI2
H3CHN ~~
(R )r--
N(Me)S(O)
R4 ~SO _
NalO4 ~N~ R 4
(R )r ~ RuCI3 ~ R
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1 9
SCHEME 3
~ HCHO, aq.
R3 R4 CH3CN, NaCNBH3
~/\NHCH3
R3 R4
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SCHEME 4
OH
SC(O)CH3
DEAD ~\N
6)~ PPh3 CH3C(O)
S(O)CI
Ac20 N~N Et~N
CH2CI2
Cocc2 ~ R3
S(O)NH~
R NalO4 ~' ~ R4
~ RoCI3
(R6)r/~J aq.
(R6)r
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-21 -
- SCHEME 5
R7 CH3 1) HNO2,Br2 7 ~CO2CH3
~ 2)KMnO
H2N N~ 3) MeOH,H+ Br N
R~MgCI R\ ~\5' CO2CH3
ZnCI2,NiCl2(Ph3p)2 N
R6 7
NaBH4 (excess) ~,CH20H
R6 7
S03 Py Et3N ~ CHO
R6 R7
CH3NH2 ~\~ \~CH2NHCH3
Na(AcO)~BH ~ ~ J
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SCHEME 6 R6
~\~Co2cH3 ~\ 9 ~"Co2cH3
Zn, CuCN
R6 R6
NaBH4 ~j~ SO3Py, Et3N ~
(excess) R~\~CH20H DMSO R\~CHO
Br~C02cH3 I~\MgCI ~ C02CH3
N ZnCI2, NiCI2(Ph3P)2 R7 ~
R6 R6
NaBH4 ¦ SO3Py, Et3N
~ CH20H ~ ~ CHO
(excess) ~N ~ DMSO ~'N
CH3NH2 ¢~
Na(AcO)3BH ~,CH2NHCH3
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SCHEMF 7 CO2CH3
Br~1. LDA, CO2 Br~
N2. MeOH, H+ N
R6
~/\MgCI ~ CH3
ZnCI2, Nicl2(ph3p)2 N~
R6
NaBH4 (excess) ~ CH20H SO3 Py, Et3N
~ R7 DMSO
R6
CHO CH3NH2 ~ CH2NHCH3
Na(AcO)3BH ~? - R7
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SCHEME 8 CO2CH3
R7 ~' 1. LDA, CO2 Br
2. (CH3)3SiCHN2
R6 ~\Br R6 ~
N ~ C02C H3
Zn, NiCI2(Ph3P)2 R7 ;~
R6 1~
excess NaBH4 l~ SO3 Py, Et3N
R7 N;~CH20H DMSO
R6 ~ CH3NH2
N~,CHO Na(AcO)3BH 7 N~
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-25 -
SCHEME 9
Boc NHl IX CH3NH2 HCI Boc NHl
Boc NH CHO NaBH(OAc)3 Boc NH CH2NHCH3
Et3N . CICH2CH2CI
/ ~
J
R3 R4
R4
B~CNHl f/'' 3
Boc NH~NcH3s(o3~
RuCI3\CF3CO2H
aq.\(~,H2CI2
Boc NH~ 1~--R3
Boc NH NCH3S02 R4 R4
CF3CO2H H2N ~ R3
CH2C12 H2N NCH3S(Ot /
~, R4
H2N ~ ~ R3
H2N N C H3S02
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SCHEME 10
BnOl CH3Nlt2-HCI
Boc NH CHONaBH(OAc)3
Et3N, CICH2CH2CI
CH2CI2
BnO ~ Et3N
Boc NH CH2NHCH3 S(O)CI
~1
R3 R4
R 4 Nal~4
BnO ~ R3 RuCI3
Boc NH NCH3S(O
.. .... . . ..
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- SCHEME 11
R4
H2N ~ ~/~/--R3 Boc20
H2N NCH3S02 CH2CI2
R4 ,~CH0
BocHN ~ ~ R3 l~J
H2N NCH3S02 NaBH(OAc)3
Et3N, CICH2CH2CI
R4
BocHN ~ 3
HN NCH3S02
~J CF3CO2H, CH2CI2
R4
H2N~ R3 ~NC
HN NCH3S02
AgCN
,~
W <N~ R3
N NCH3S02
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-2~ -
SCHEME 12
~4
BnO ~ ~R 20% Pd(~H)2~ H2
~ ~ CH30H
Boc NH NCH3S02 CH3CO2H
R4
HO ~/~ R3
') CICOCOCI
Boc NH --NCH3S02--~/ DMSO CH2C12
(C2H5)3N
R4 1. R'MgX
H~O ~/~R3 (C2Hs)2~
~ 2 TFA
Boc NH NCH3S02 CH2C12
,~4
R, OH ~ ~R3
H2N NC H3S02
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-29 -
SCHEME 13
H0~ ~ R3 CF3CO2H
Boc NH NCH3S02 CH2CI2
R4
H0 ~, R R'CH0
H2N~--NCH3S0-- NaBH(OAc)3
CICH2CH2CI
R4
H0 ~/';\ R3
R'C H2N H NC H3S02
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SCHEME 14
N
R4 N=\N~ ,N/=
HO~ ~/~ R3 ~ O
Boc NH NCH3S02 NaH, DMF 0~C
R4
~/~R3 R'SH
~ (c2H5)3
Boc N --NCH3S02 CH30H
R4
R'S ~ ~ HCI, EtOAc
Boc NH NCH3S02
R'S~ ~R3
H2N NCH3S02
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SCHEME 15
HO~ 1) Boc20, K2C~3 HO~
,~/ THF-H20 ~
2) C H2N EtOAc
H2N CO2H 2, BocNH CO2CH3
XXVI I
XXVIII
HO~
LiAlH4 ~JJ R'CH2X
THF ~ Cs2CO3
0-20~C BocNH CH2OH DMF
XXIX
R 'C H20~ R 'C H20~
,~W OMSO ,~lV
BocNH CH2OH (C2Hs)3N BocNH CHO
20~C
XXX XXXI
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SCHEME 16
R'CH ~3 CH3NH2-HCI RCH20
Na(OAc)3BH
BocNH CHO CICH2CH2CI BocNH/~ NHCH3
CH2CI2 /
~S(O)CI
R3 R4
R'CH
BocNH NCH3S(O
NalO4\
RuCI3 \~
aq.
R'CH~,,~ 3 ~R4
BocNH NCH3s02
~/~ 20% Pd(OH)2 \
CH30H, CH3C02H\ EHtCOA
2. HCI, EtOAC
R'CH~O~ r~,R4
H2N NCH3S02 H2N NCH3S02
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The instant compounds are useful in the treatment of
cancer. Cancer.s which may be treated with the compound,s of thi~
invention include, but are not limited to, colorectal carcinoma,
exocrine pancreatic carcinoma, myeloid leukemia,s and neurological
tumors. Such tumors may arise by mutation~ in the ra~ genes
themselves, mutation.s in the proteins that can l-egulate Ra,s activity (i.e.,
neurofibromin (NF-l), neu, ,scr, abl, Ick, fyn) or by other mechanism,~.
The compound,~i of the in~itant invention inhibit farne,syl-
protein tr~n~sferase and tarne~ylation of the oncogene protein Ra,s.
The instant compounds may al,so inhibit tumor angiogenelsi,~, thereby
affecting the growth of tumors (J. Rak et al. Cfln( e~- Re~earch, 55:4575-
45~0 (1995)). Such anti-~ngiogenic propertie,s of the in~tant compounds
may also be u~eful in the treatment of certain form~s of blindnes,<i related
to retinal vascularization.
The compounds of this invention are also u,seful for
inhibiting other diseases where Ras protein,s are aberrantly activated as
a result of oncogenic mutation in other gene,s (i.e., the Ra,s gene itself i~
not activated by mutation to an oncogenic form) with said inhibition
being accomplished by the administration of an effective amount of the
compound,s of the invention to a mammal in need of ,such treatment.
For example, a component of NF-I is a benign proliferative disorder.
The instant compounds may also be u~eful in the treatment
of viral infections, in particular in the treatment of hepatiti,s delta and
related viru,se,s (J.S. Glenn et al. Scienef~, 256: 1331 - 1333 (1992).
The compounds of the in~tant invention are al,so useful in
the prevention of resteno,~ after percutaneou,s tran,sluminal coronary
angioplasty by inhibiting neointimal formation (C. Indolfi et al. Natu~ e
medicine, 1 :541-545(1995).
The instant compounds may al~o be useful in the treatment
and prevention of polycy,stic kidney disease (D.L. Schaffner et al.
American J(~urnal (~f Path(~lo(~y~ 142:1051-1060 (1993) and B. Cowley,
Jr. et al.FASEB Jf~urnal, 2:A3160 (19~
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The instant compounds may also be useful for the treatment
of fungal infections.
The compounds of this invention may be administered to
m;lmm~l~;, preferably humans, either alone or, preferably, in combina-
S 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.
The compound.s can be admini~stered orally, topically, rectally, vaginally
transdermally or parenterally, including the intravenous, intramuscular,
10 intraperitoneal and subcutaneous routes of administration.
For oral use, the compound is administered, for example,
in the form of tablets or capsules, or as a solution or ~suspension. ln the
case of tablets for oral use, carrier~s which are commonly used include
lactose and corn starch; lubricating agents, such as magnesium stearate,
15 are commonly added. For~ral administration in capsule form, diluents
also include lactose and dried corn starch. When a4ueou.s 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, intra-
20 peritoneal, subcutaneous and intravenous use, sterile solution~s of the
active ingredient are usually prepared, the pH of the solution is ~suitably
adjusted and the product is buffered. For intravenous use, the total
concentration is controlled to render the preparation substantially
isotonic.
The compounds of the instant invention may also be
co-:~(lministered with other well known therapeutic agents that are
selected for their particular usefulness against the condition that is
being treated. For example, the instant compounds may be useful in
combination with known anti-cancer and cytotoxic agents. Similarly,
the instant compounds may be useful in combination with agents that are
effective in the treatment and prevention of NF-I, restinosis, polycystic
kidney disease, infections of hepatitis delta and related viruses and
fungal infections.
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If formulated as a fixed dose, such combination product,s
employ a compound of this invention substantially within the dosage
range de,scribed below and other pharmaceutically active agent(s)
typically within the acceptable do,sage range. Compounds of the instant
S invention may alternatively be u,sed se(Juentially with known pharma-
ceutically acceptable agent(,s) when a combination forrnulation is
inappropriate.
The daily dosage will normally be determined by the
prescribing physician, who may vary the do,sage according to the age,
10 weight, and respon,se of the individual patient, as well as the severity of
the patient'~s condition.
In one exemplary application, a suitable amount of
compound i.s administered to a m~mm~l undergoing treatment for
cancer. Admini~stration occurs in an amount between about 0.1 mg/kg
15 of body weight to about 60 mg/kg of body weight per day, preferably of
between 0.5 mg/kg of body weight to about 40 mg/kg of body weight
per day.
The compound,s of the instant invention are also u~seful
as a component in an assay to rapidly determine the pre~sence and
20 quantity of farne.syl-protein transferase (FPTa,se) in a composition.
Thu.s 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 farnelsyl pyrophosphate and, in one of the mixture~s,
25 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-
logical, radiochemical or chromato~raphic technique~. Because
30 the compounds of the instant invention are selective inhibitors of
FPTa~se, ab.sence or quantitative reduction of the amount of substrate
in the a~s~say mixture without the compound of the instant invention
relative to the prelsence of the unchanged ~sub~strate in the assay
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containing the instant compound is indicative of the presence of
FPTa,se in the composition to be tested.
It would be readily apparent to one of ordinary skill in the
art that ~uch an assay as described above would be useful in identifying
5 tissue sample,s which contain farne~yl-protein tran~ferase and quanti-
tating the enzyme. Thus, potent inhibitor compounds of the instant
invention may be used in an active site titration assay to determine the
quantity of enzyme in the sample. A ~erie~ of !;ample~i composed of
aliquots of a tissue extract containing an unknown amount of farne,syl-
10 protein tran~sferase, an exces~; amount of a known substrate of FPTa~ie
(for example a tetrapeptide havin~ a cy~teine at the amine terrninus) 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
lS that ha.s a Ki substantially ~smaller than the concentration of enzyme in
the as~;ay 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
N~ (4-CYANOBENZYL)IMIDAZOLYL-5-METHYLl-N-
(METHYL)-3 -CHLOROBENZYLSULFONAMIDE
HYDROCHLORIDE
Step A: Preparation of l-triphenylmethyl-4-~hydroxymethyl)imidazole
To a .~olution of 4-(hydroxymethyl)imidazole
hydrochloride (35.0 g, 260 mmol) in 250 mL of dry DMF at room
temperature wa~ added triethylamine (90.6 mL, 6~0 mmol). A white
30 solid precipitated from the solution. Chlorotriphenylmethane (76.1 ~,
273 mmol) in 500 mL of DMF wa~i added dropwi~ie. The reaction
mixture wa.s ,stirred for 20 hour~s, poured over ice, filtered, and wa~;hed
with ice water. The resulting product wa.~ ~lurried with cold dioxane,
filtered, and dried i)1 V~ 'UO to provide the titled product a~ ~ white solid
35 which wa~ I~ufficiently pure for u~se in the next .step.
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Step B: Preparation of 1 -triphenylmethyl-4-(acetoxvmethyl)imidazole
Alcohol from Step A (260 mmol, prepared above) wa~
su,spended in 500 mL of pyridine. Acetic anhydride (74 mL, 780
S mmol) wa,s added dropwi.se, and the reaction wa,s ,stirred for 4~ hours
during which it became homogeneous. The solution was poured into 2
L of EtOAc, washed with water (3 x 1 L), 5% a~. HCl ,soln. (2 x 1 L),
,sat. aq. NaHCO3, and brine, then dried (Na2SO4), filtered, and
concentrated in va~o to provide the crude product. The acetate wa~
10 ilsolated as a white powder (g5.X g, ~6% yield for two .step.s) which wa~
.sufficiently pure for use in the next reaction.
Step C: Preparation of 1-(4-cyanobenzyl)-5-(acetoxymethyl)imidazole
hydrobromide
A ~solution of the product from Step B (~5.X g, 225
mmol) and ~-bromo-p-tolunitrile (50.1 g, 232 mmol) in 500 mL of
EtOAc wa~i stirred at 60~C for 20 hour~s, 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 wa,s
20 concentrated in vac~u~ to a volume 200 mL, reheated at 60~C for two
hour.s, cooled to room temperature, and filtered again. The filtrate
was concentrated in vac~uo 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 Isolid material was
25 combined, dissolved in 500 mL of methanol, and walmed to 60 ~C.
After two hours, the solution wa,s reconcentrated in vacuo to provide
a white lsolid which wa,s triturated with hexane to remove .soluble
material,s. Removal of re,sidual ,solvents il~ vacu~ provided the titled
product hydrobromide a~s a white solid (50.4 g, 67~/~, yield, ~9% purity
30 by HPLC) which wa~ u,sed in the next ,step without further purification.
Step D: Preparation of 1-(4-cyanobenzyl)-5-(hydroxymethvl)imidazole
To a ~;olution of the acetate from Step C (50.4 ~, 150
mmol) in 1.5 L of 3:1 THF/water at 0 ~C wa,s added lithium hydroxide
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-3g-
monohydrate (18.9 g, 450 mmol). After one hour, the reaction was
concentrated in l~acuo, 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 (26.2 g, 82% yield) as a pale yellow fluffy solid which was
sufficiently pure for use in the next .step without further purification.
Step E: Preparation of 1-(4-cyanobenzyl)-5-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 triethyl-
amine (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 vacMo to provide the aldehyde (1 ~.7 g, ~X% 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-
I (methylamino)methyllimidazole
To a suspension of methylamine hyd~ochloride in 5 mL
dichloroethane at 0~C are added 4 A sieves (0.5g), followed by 1 mmol
of the aldehyde from Step E and 1.5 mmol Na(OAc)3BH. The reaction
is stirred for 10 minutes at 0~C, warmed to room temperature and
stirred for 2 hours. The reaction i~s poured into EtOAc/ ~sat. NaHCO3
solution. The organic layer i~s washed with brine, dried (Na2SO4 ),
filtered, and concentrated in vacuo. The crude product is taken up in
10 mL dichloromethane and 2 mL n-propylamine, stirred at room
temperature for I hour, concentrated in vacuo, and purified by flash
chromatography .
Step G: Preparation of 3-chlorobenzyl thioacetate
To a solution of 1 mmol 3-chlorobenzyl alcohol and
I mmol triphenylpholsphine in 5 mL THF at 0~ C, I mmol diethyl-
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-39-
azodicarboxylate is added. After stirring for 10 minutes, I mmol
thioacetic acid is added. The reaction i~s stirred for 3 hours,
concentrated and purified by flash chromatography.
StepH: Preparation of 3-chlorobenzyl.sulfinyl ch~oride
To a -20~ C solution of the thioester from Step G (1 mmol)
in 3 mL of dichloromethane under ar~on are added I mmol acetic
anhydride and 2 mmol sulfuryl chloride. The reaction is stirred for I
hr during which time the temperature is allowed to rise to -5~ C. The
mixture is concentrated in ~~ac~uo and the crude product used for
coupling in the next step.
Step I: Preparation of N-[1-(4-cyanobenzyl)imidazolyl-~-methyl]-
N-(methyl)-3 -chlorobenzylsulfinamide
To a 0~ C solution of the sulfinyl chloride from Step H
(I mmol) in 4 mL dichloromethane i,s added a solution of 1 mmol
triethylamine and I mmol of the amine from Step F in 2 mL dichloro-
methane. The reaction i,~ stirred overnight and the temperature is
allowed to rise to room temperature. This mixture is poured into ethyl
acetate, washed with sat. NaHCO3 solution and brine, dried with
Na2SO4, concentrated and purified by fla,sh chromatography.
Step J: Preparation of N-[ 1 -(4-cyanobenzyl)imidazolyl-5-methyl] -
N-(methyl)-3-chlorobenzylsulfonamide hydrochloride
A solution of the ,sulfinamide from Step I (1 mmol) in
2 mL acetonitrile is cooled to 0~C. NaIO4 i,~i added (1.5 mmol)
followed by a catalytic amount of RuCI~ 3H~O and 2 mL H2O. The
reaction is stirred at room temperature for 1 hour, diluted with EtOAc,
and washed with ,sat. NaHCO3 solution and brine, dried (Na2SO4),
filtered and concentrated. The resulting product is purified by flash
chromatography~ then taken up in dichloromethane, and treated with
with excess ethereal HCI. Concentration in ~ UO provides the titled
product.
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EXAMPLE 2
N-(3-CHLOROBENZYL)-1 -I (4-CYANOBENZYL)-5-
IMIDAZOLYLlMETHYLSULFONAMIDE HYDROCHLORIDE
Step A: Preparation of 1-(4-chlorobenzyl)-5-imidazolylmethyl
sulfinyl chloride
The titled compound i.s prepared from the alcohol from
Step D of Example 1 by the method.s described in Steps G and H of
Example 1.
Step B: Preparation of N-(3-chlorobenzyl)-1-L(4-cyanobenzyl)-5-
imidazolyllmethylsulfinamide
The sulinyl chloride from Step A i.s coupled with 3-
chlorobenzylamine using the method de~icribed in Step I of Example 1.
Step C: Preparation of N-(3-chlorobenzyl)- 1 -[(4-cyanobenzyl)-5-
imidazolyllmethylsulfonamide hydrochloride
The sulfinamide from Step B is oxidized to the sulfonamide
and converted to the HCI ~alt using the method delscribed in Step J of
Example 1.
EXAMPLE 3
N-l 3-(4-CYANOBENZYL)PYRIDYL-4-METHYLl-N-(METHYL)-3-
CHLOROBENZYLSULFONAMIDE HYDROCHLORIDE
Step A: Preparation of 3-(4-cyanobenzyl)pyridin-4-carboxylic acid
methyl ester
A solution of 4-cyanobenzyl bromide (625 mg, 3.27 mmol)
in dry THF (4mL) wa,s 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 wa~s removed and the ,slurry wa~i ,stirred at
room temperature for a further 30 min. Then 3-bromopyridin-4-
carboxylic acid methyl ester (540 mg. 2.5 mmol) followed by
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dichlorobis(triphenylphosphine)nickel (II) (50 mg). The resultant
reddish-brown mixture was stirred for 3h at ~40-45~C. The mixture
was cooled and distributed between EtOAc (100 ml) and 5% a4ueous
citric acid (50 mL). The organic layer was washed with H2O (2XS0
5 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 ~odium borohydride
(300 mg) reduction of the ester from Step A (41~ mg) in methanol
(5 mL) at room temperature. After stirring for 4 h the .~;olution was
evaporated and the product wa~i purified on silica gel, eluting with 2%
methanol in chloroform to give the title compound. FAB m~; (M+l)
1 5 225.
Step C: Preparation of 3-(4-cyanobenzyl)-4-pyridinal
The title compound was obtained by activated manganese
dioxide (l.Og) oxidation of the alcohol from Step B (240 mg, 1.07
mmol) in dioxane (10 mL) at reflux for 30 mim Filtration and
evaporation of the ~olvent provided title compound, mp ~0-~3~C.
Step D: Preparation of 3-(4-cyanobenzyl)-4-
I (meth ylamino)methyl lpyridine
The titled compound i~; prepared from the pyridinal from
Step C and methylamine hydrochloride using the procedure in Step F of
Example 1.
Step E: Preparation of N-[3-(4-cyanobenzyl)pyridyl-4-methyl]-N-
(methyl)-3-chlorobenzyl~ulfon~mide hydrochloride
The titled compound i~ prepared from the amine from Step
D and the sulfinyl chloride from Step H of Example I u~iing the
procedure.s de.scribed in Step.~; I and J of Example 1.
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ln vitro inhibition of ra.s farnesyl tran~sferase
Assays of farnesyl-protein t~ aMsfe7 ase~ Partially purified
bovine FPTase and Ras peptides (Ras-CVLS, Ras-CVIM and Ras-CAIL)
were prepared as described by Schaber et al., J. Biol. Chem. 265:14701-
14704 (1990), Pompliano, et al., Biochemistry 31:3~00 (1992) and
Gibbs et ah, PNAS U.S.A. ~6:6630-6634 (1989), respectively. Bovine
FPTa,se was assayed in a volume of 100 ~I cont~ining 100 mM N-(2-
hydroxy ethyl) piperazine-N'-(2-ethane .sulfonic acid) (HEPES), pH
7.4, 5 mM MgC12, 5 mM dithiothreitol (DTT), 100 mM ~3H]-farne.syl
diphosphate ([3H]-FPP; 740 CB4/mmol, New England Nuclear), 650 nM
Ras-CVLS and 10 ,ug/ml FPTase at 31 ~C for 60 min. Reactions were
initiated with FPTa.se and stopped with 1 ml of 1.0 M HCL in ethanol.
Precipitates were collected onto filter-mat~; using a TomTec Mach II cell
harvestor, washed with 100% ethanol, dried and counted
in an LKB ,B-plate counter. The assay wals linear with re.spect to both
substrates, FPTase levels and time; less than 10% of the [3H]-FPP wa.s
utilized during the reaction period. Purified compound~ were dis.solved
in 100% dimethyl sulfoxide (DMSO) and were diluted 20-fold into
the assay. Percentage inhibition is mea.sured by the amount of
incorporation of radioactivity in the presence of the te.st compound
when compared to the amount of incorporation in the ab.sence of
the test compound.
Human FPTase was prepared as described by Omer
et al., Biochemistry 32:5167-5~76 (1993). Human FPTase activity
was assayed as described above with the exception that 0.1 % (w/v)
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 a.s described above for the bovine enzyme.
n vil~o ra.s farne!iylation assay
The cell line used in this a.ssay is a v-ras line derived
from either Ratl or NIH3T3 cells, which expressed viral Ha-ras p21.
The assay is performed e~sentially as de.scribed in DeClue, J.E. et al.,
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WO 97/36583 PCT/US97/05170
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Cancer Research 51 :712-717, (1991). Cells in 10 cm dishe.s at 50-75%
confluency are treated with the te,st 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-
5 meted with 10% regular DMEM, 2% fetal bovine serum and 400mCi[35S]methionine (1000 Ci/mmol). After an additional 20 hours, the
cells are Iysed in 1 ml Iy.sis 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) arld the Iysates cleared by centrifugation at
100,000 x g for 45 min. Aliquot.s of Iysate,s containing equal numbers
of acid-precipitable count.s are bought to 1 ml with IP buffer (Iysis
buffer lacking DTT) and immunoprecipitated with the ras-specific
monoclonal antibody Y13-259 (Furth, M.E. et al., J. Virol. 43:294-304,
(19~2)). Following a 2 hour antibody incubation at 4~C, 200 ml of a
15 25% suspension of protein A-Sepharo.se coated with rabbit anti rat IgG
is added for 45 min. The immunoprecipitate,s are wa~shed four times
with IP buffer (20 nM HEPES, pH 7.5/1 mM EDTA/I % 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
20 reached the bottom, the gel is fixed, soaked in Enlightening, dried and
autoradiographed. The intensities of the band,s corre,sponding to
farne,sylated and nonfarnesylated ra,s proteins are compared to
determine the percent inhibition of farnesyl tran,sfer to protein.
25 In Vil)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 cell,s transformed with either a
v-ras, v-7 af, or v-mos oncogene is te,sted. Cells transformed by v-Raf
30 and v-Mos maybe included in the analysi,s to evaluate the ,specificity of
instant compounds for Ras-induced cell transformation.
Rat 1 cells tran,sformed with either v-ras, v-raf, or v-mo~
are ~eeded at a density of 1 x 104 cell.s per plate (3~ mm in diameter) in
a 0.3% top agarose layer in medium A (Dulbecco'~ modified Eagle's
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medium supplemented with 10% fetal bovine .serum) over a bottom
agaro~ie layer (0.6%). Both layers contain 0.1% methanol or an
appropriate concentration of the in.stant compound (dissolved in
methanol at 1000 times the final concentration used in the assay). The
S cells are fed twice weekly with 05 ml of medium A containing 0.1%
methanol or the concentration of the instant compound. Photomicro-
graphs are taken 16 days after the cultures are seeded and comparisons
are made.
