Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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TITLE OF THE ~NVENTION
INHIBITORS OF FARNESYL-PROTElN TRANSFERASE
BACKGROUND OF THE INVENTION
S The Ras proteins (Ha-Ras, Ki4a-Ras, Ki4b-Ras and
N-Ras) are part of a signaling pathway that links cell surface growth
factor receptors to nuclear signals initiating cellular proliferation.
Biological and biochemical studies of Ras action indicate that Ras
functions like a G-regulatory protein. Ln the inactive state, Ras is bound to
GDP. Upon growth factor receptor activation Ras i,s 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 Ras, which returns the
protein to its inactive GDP bound form (D.R. Lowy and D.M. Willumsen,
Ann. Rev. Biochem. 62:851-~91 (1993)). Mutated ~as genes (Ha-ras,
Ki4a-ras, Ki4b-ras and N-ras) are found in many human cancers,
including colorectal carcinoma, exocrine pancreatic carcinoma, and
myeloid leukemias. The protein products of these genes are defective in
their GTPase activity and constitutively transmit a growth stimulatory
signal.
Ras must be localized to the plasma membrane for
both normal and oncogenic functions. At least 3 post-translational
modifications are involved with Ras membrane localization, and all
3 modifications occur at the C-terminus of Ra.s. The Ras C-terminus
contains a sequence motif termed a "CAAX" or "Cys-Aaal-Aaa2-Xaa"
box (Cy.s is cysteine, Aaa is an aliphatic amino acid, the Xaa is any amino
acid) (Willumsen et al., Natu~ e 310:5~3-5~6 ( 19~4)). Depending on the
specific sequence, this motif serves as a signal sequence for the enzymes
farnesyl-protein transferase or geranylgeranyl-protein transferase, which
catalyze the alkylation of the cysteine residue of the CAAX motif with a
Cls or C20 isoprenoid, re.spectively. (S. Clarke., Ann. Rev. Biochem.
61:355-3~6 (1992); W.R. Schafer and J. Rine, Ann. Rel~. Geneties 30:209-
237 ( 1992)). The Ras protein is one of several proteins that are known to
undergo post-translational farnesylation. Other farnesylated protein.s
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include the Ras-related GTP-binding proteins such as Rho, fungal mating
factors, the nuclear lamins, and the gamma subunit of transducin. James,
et al., J. Biol. Chem. 269, 141X2 (1994) have identified a peroxisome
associated protein Pxf which is also farnesylated. James, et al., have also
5 suggested that there are farnesylated proteins
of unknown structure and function in addition to those listed above.
Inhibition of farnesyl-protein transferase has been shown
to block the growth of Ras-transformed cells in soft agar and to modify
other aspects of their transformed phenotype. It has also been demon-
10 strated that certain inhibitors of farnesyl-protein transferase selectively
block the processing of the Ras oncoprotein intracellularly (N.E. Kohl
et al., Scienc~e, 260:1934-1937 (1993) and G.L. James et al., Science,
260:1937-1942 (1993). Recently, it has been shown that an inhibitor of
farnesyl-protein transferase blocks the growth of ras-dependent tumors in
15 nude mice (N.E. Kohl et al., Proc. Natl. Acad. Sci U.S.A., 91:9141-9145
(1994) and induces regression of m~mm~ry and salivary carcinomas in
ras transgenic mice (N.E. Kohl et al., Nature Medicine, 1 :792-797
(1 995).
Indirect inhibition of farnesyl-protein transferase in l ivo
20 has been demonstrated with lovastatin (Merck & Co., Rahway, NJ)
and compactin (Hancock et al., ibid; Casey et al., ibid; Schafer et al.,
Science 245:379 (1989)). These drugs inhibit HMG-CoA reductase, the
rate limiting enzyme for the production of polyisoprenoids including
farnesyl pyrophosphate. Farnesyl-protein transferase utilizes farnesyl
25 pyrophosphate to covalently modify the Cys thiol group of the Ras
CAAX box with a farnesyl group (Reiss et al., Cell, 62:81-88 (1990);
Schaber et al., J. Biol. Chem., 265: 14701 -14704 (1990); Schafer et al.,
Science, 249: 1133- 1139 (1990); Manne et ~1., Proc . Natl. Acad. Sci USA.
87:7541 -7545 (1990)). Inhibition of farnesyl pyrophosphate biosynthesis
30 by inhibiting HMG-CoA reductase blocks Ras membrane localization in
cultured cells. However, direct inhibition of farnesyl-protein transferase
would be more specific and attended by fewer side effects than would
occur with the required dose of a general inhibitor of isoprene
biosynthesis.
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Inhibitors of farnesyl-protein transferase (FPTase) have
been described in two general classes. The fir~st are analogs of farnesyl
diphosphate (FPP), while the second class of inhibitors is related to
the protein substrates (e.g., Ras) for the enzyme. The peptide derived
S inhibitors that have been described are generally cysteine containing
molecules that are related to the CAAX motif that is the signal for protein
prenylation. (Schaber et al., ibid; Reis.s et. al., ihid; Reiss et al., PNAS,
88:732-736 (1991)). Such inhibitors may inhibit protein prenylation
while serving as alternate substrates for the farnesyl-protein transferase
10 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)). In general, deletion of
the thiol from a CAAX derivative has been shown to dramatically reduce
the inhibitory potency of the compound. However, the thiol group
15 potentially places limitations on the therapeutic application of FPTase
inhibitors with respect to pharmacokinetics, pharmacodynamics and
toxicity. Therefore, a functional replacement for the thiol is desirable.
It has recently been reported that farnesyl-protein tran.sferase
inhibitors are inhibitor.s of proliferation of vascular smooth muscle cell.s
20 and are therefore useful in the prevention and therapy of arteriosclero,sis
and diabetic disturbance of blood vessels (JP ~7- 112930).
It has recently been disclosed that certain tricyclic
compounds which optionally incorporate a piperidine moiety are
inhibitors of FPTase (WO 95/10514, WO 95/10515 and WO 95/10516).
25 Imidazole-containing inhibitors of farnesyl protein transferase have also
been disclosed (WO 95/09001 and EP 0 675 112 Al ).
It is, therefore, an object of this invention to develop
peptidomimetic compounds that do not have a thiol moiety, and that
will inhibit farnesyl-protein transferase and thus, the post-translational
30 farnesylation of proteins. It is a further object of this invention to develop
chemotherapeutic compo,sitions containing the compounds of this
invention and methods for producing the compounds of this invention.
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SUMMARY OF THE INVENTION
The present invention comprises analogs of the CA1A2X
motif of the protein Ras that is modified by farnesylation in vivo. These
CA 1 A2X analogs inhibit the farnesylprotein transferase. Furthermore,
5 these CA 1 A2X analogs differ from those previously described as
inhibitors of farnesyl-protein transferase in that they do not have a thiol
moiety. The lack of the thiol offers unique advantages in terms of
improved pharmacokinetic behavior in ~nim~ , prevention of thiol-
dependent chemical reactions, such as rapid autoxidation and disulfide
10 formation with endogenous thiols, and reduced systemic toxicity. The
compounds of the instant invention also incorporate a cyclic amine
moiety in the A2 position of the motif. The compounds of the instant
invention also do not contain a carboxylic acid, and therefore do not
require a prodrug ester for improved cell permeability. Further contained
15 in this invention are chemotherapeutic compositions containing these
farnesyl transferase inhibitors and methods for their production.
The compounds of this invention are illustrated by the
formulae:
(R8) /~9\ Z
V ~ A1(CR1az)nA2(cR1 2)n -\w/ - (CRlb2)p~N~N
DETAILED DESCRIPTION OF THE INVENTION
The compounds of this invention inhibit the farnesyl-protein
25 transfera.se. In a first embodiment of this invention, the farnesyl-protein
transferase inhibitors are illustrated by the formula 1:
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(R8)r /R9\ Z
V - A1 (C R1 a2)nA2(C R l a2) n ~W/ - (C R l b2)~ N~N~RRs5b
R4b
R4a
wherein:
Rla and R1b are independently selected from:
a) hydrogen,
b) aryl, heterocycle, cycloalkyl, alkenyl, alkynyl,
R10O-, Rl 1S(o)m, R10C(O)NRl0-~ CN, NO2,
(R 1 0)2N-C(NR 10) , R 1 ~C(O)-, CON(R 1~)2-, N3,
-N(R10)2, or Rl lOC(O)NR10-
c) Cl-C6 alkyl unsubstituted or substituted by aryl,
heterocyclic, cycloalkyl, alkenyl, alkynyl, R100-,
Rl 1S(O)m-, R10C(o)NR10-, CN, (R10)2N-C(NR10)-,
R10C(o)-, CON(R10)2-, N3, -N(R10)2, or Rl loC(O)-
NR10-;
R2 and R3 are independently selected from:
a) a side chain of a naturally occurring amino acid,
b) an oxidized form of a side chain of a naturally occurring
amino acid which is:
i) methionine sulfoxide, or
ii) methionine sulfone, and
c) substituted or unsubstituted C 1 -C20 alkyl, C2-C20 alkenyl,
C3-Clo cycloalkyl, aryl or heterocyclic group,
wherein the substituent is selected from F, Cl, Br,
N(R 1~)2, NO2, R 10o-, R 1 1 S(O)m ~ R 1 0C(o)NR 10,
CN, (R 10)2N-C(NR 10), R10c(o)-~ CON(R10)2-,
N3, -N(R10)2, Rl lOC(O)NR10- and Cl-C20 alkyl,
and
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d) Cl-C6 alkyl substituted with an unsubstituted or
substituted group selected from aryl, heterocycle and
C3-CIo cycloalkyl; or
S R2 and R3 are combined to form - (CH2)S -; or
R2 or R3 are combined with R6 to form a ring such that
R6 ~
~; N ~ is ~ H2)t ;
R2 R3 R7a~R7b
R4a, R4b, R7a and R7b are independently selected from:
a) hydrogen,
b) C1 -C6 alkyl unsubstituted or substituted by alkenyl, R 10o-,
Rl lS(O)m-, RlOC(O)NR10-, CN, N3, (R10)2N-C(NlR10)-,
R1OC(O)-, CON(R10)2-, -N(R10)2, or Rl lOC(O)NR10-,
c) aryl, heterocycle, cycloalkyl, alkenyl,
RlOO, Rl lS(o)m, Rloc(o)NRlo-~ CN, NO2,
1 0)2N C(NR 10), R 1 ~C(O)-, CON(R 1~)2-, N3,
-N(R10)2, or Rl lOC(O)NR10-, and
d) Cl-C6 alkyl substituted with an unsubstituted or
substituted group selected from aryl, heterocyclic and
C3-Clo cycloalkyl;
R5a and R5b are independently selected from:
a) hydrogen,
b) substituted or unsubstituted C 1 -C20 alkyl, C2-C20 alkenyl,
C3-Clo cycloalkyl, aryl or heterocycle group,
wherein the substituent is selected from F, Cl, Br,
(R 1 0)2NC(O)-, NO2, R l Oo ~ R l l S(o)m
R 1 OC(O)NR 10, CN, (R 1 0)2N-C(NR 1 0) , R 1 ~C(O)-,
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CON(R 1 0)2-? N3, -N(R 1 0)2, R l l OC(O)NR 1 0- and
Cl-C20 alkyl,
d) Cl-C6 alkyl sub,stituted with an un~substituted or
substituted group selected from aryl, heterocycle and
C3-CIo cycloalkyl; or
RSa and RSb are combined to form - (CH2)S - wherein one of the
carbon atoms is optionally replaced by a moiety selected from:
O, S(O)m, -NC(O)-, and -N(COR10)-;
R6 is independently selected from hydrogen or Cl-C6 alkyl;
R~ is independently selected from:
a) hydrogen,
b) aryl, heterocycle, cycloalkyl, alkenyl, alkynyl,
perfluoroalkyl, F, Cl, Br, R10O-, Rl lS(O)m-,
R 1 0C(O)NR 10, CN, NO2, R 1 02N-C(NR 10) , R l ~C(O)-,
CON(R10)2-, N3, -N(R10)2, or Rl 1OC(O)NR10-, and
c) Cl-C6 alkyl unsubstituted or substituted by aryl,
heterocycle, cycloalkyl, alkenyl, alkynyl, perfluoroalkyl,
F, Cl, Br, R10O-, Rl lS(O)m-, R10C(O)NH-, CN,
H2N-C(NH)-, R10C(o)-, CON(R10)2-, N3, -N(R10)2, or
CON(R 1 0)2NH-;
25 R9 is selected from:
a) hydrogen,
b) alkenyl, alkynyl, perfluoroalkyl, F, Cl, Br,
R10O-, Rl lS(o)m, R10C(O)NR10-~ CN, NO2,
(R 1 0)2N-C-(NR 10), R 1 ~C(O)-, CON(R 1~)2-, N3,
-N(R 1 0)2, or R l l OC(O)NR 1 0-, and
c) Cl-C6 alkyl unsubstituted or ,substituted by perfluoroalkyl,
F, Cl, Br, R10O-, RllS(O)m-, R10C(o)NR10-, CN,
(R10)2N-C(NR10)-, R10C(O)-, CON(R10)2-, N3,
-N(R 1~)2, or R 1 1 OC(O)NR 10;
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_ " _
R10 i,s independently selected from hydrogen, C1-C6 alkyl, benzyl and
aryl;
5 Rl 1 is independently selected from Cl-C6 alkyl and aryl;
A 1 and A2 are independently selected from: a bond, -CH=CH-, -C-C-,
-C(0)-, -C(O)NR 10, -NR 1 ~C(0)-, 0, -N(R 10),
-S(0)2N(R10)-, -N(RlO)S(0)2-~ or S(O)m;
Q i,s a substituted or uulsubstituted nitrogen-cont~ining C4-Cg mono orbicyclic ring system, wherein the non-nitrogen containing ring may be a
Cs-C7 saturated ring;
15 V is selected from:
a) hydrogen,
b) heterocycle,
c) aryl,
d) C1-C20 alkyl wherein from 0 to 4 carbon atoms are replaced
with a heteroatom selected from 0, S, and N, and
e) C2-C20 alkenyl,
provided that V is not hydrogen if A 1 is S(0)m and V is not hydrogen if
Al is a bond, n is 0 and A2 is S(O)m;
25 W is a heterocycle;
X, Y and Z are independently H2 or 0;
m is 0, 1 or 2;
nis 0,1,2,30r4;
pis 0, 1,2,30r4;
r is 0 to 5, provided that r is 0 when V is hydrogen;
s is 4 or S;
tis 3,40rS; and
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uis Oor l;
or the pharmaceutically acceptable salts, crystal forms, hydrates and
isomers thereof.
In a more preferred embodiment of this invention, the Ras
S farnesyl transferase inhibitors are illustrated by the Fo~nula I:
(R8)r /R9\ Z
V - A1(Cl~l ~)nA~(CR1a2)n -\w/ - (CR1b2)~
wherem:
Rla is independently selected from: hydrogen or Cl-C6 alkyl;
Rlb is independently selected from:
a) hydrogen,
b) aryl, heterocycle, cycloalkyl, RlOo, -N(R 1~)2 or alkenyl,
c) Cl-C6 alkyl unsubstituted or substituted by aryl,
heterocycle, cycloalkyl, alkenyl, R100-, or -N(R10)2;
R2 and R3 are independently selected from:
a) a side chain of a naturally occurring amino acid,
b) an oxidized form of a side chain of a naturally occurring
amino acid which is:
i) methionine sulfoxide, or
ii) methionine sulfone,
c) substituted or unsubstituted Cl-Clo alkyl, C2-Clo alkenyl,
C3-Clo cycloalkyl, aryl or heterocyclic group,
wherein the substituent is selected from F, Cl, Br,
N02, R 1 00-, R l l S(O)m-, R l OC(O)NR 10-, CN,
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- 10-
(R 1 0)2N-C(NR 10) , R 1 ~C(O)-, CON(R 1~)2-, N3,
-N(R10)2, Rl IOC(o)NRlo- and Cl-C20 alkyl, and
d) Cl-C6 alkyl substituted with an unsubstituted or
substituted group selected from aryl, heterocycle and
C3-C1 o cycloalkyl; or
R2 and R3 are combined to form - (CH2),s -; or
R2 or R3 are combined with R6 to form a ring such that
R6 R7a~R7b;
R4a and R7a are independently selected from:
a) hydrogen,
l~S b) Cl-C6 alkyl unsubstituted or substituted by alkenyl, R100-,
R1 1S(O)m-, R10C(o)NR10-, CN, N3, (R10)2N-C(NR10)-,
R 1 0c(O)-, CON(R 1 0)2-, -N(R 1~)2, or R l 1 OC(O)NR 1 0-,
c) aryl, heterocycle, cycloalkyl, alkenyl, R10O-,
R 1 1 S(O)m-~ R 1 0C(O)NR 10, CN, NO2,
(R10)2N C(NR10), R10C(o), CON(R10)2-, N3,
-N(R 1~)2, or R 1 1 OC(O)NR 10, and
d) Cl -C6 alkyl substituted with an unsubstituted or
substituted group selected from aryl, heterocyclic and
C3-Clo cycloalkyl;
R4b and R7b are hydrogen;
RSa is selected from:
a) substituted or unsubstituted C 1 -C 10 alkyl, C2-C1 0 alkenyl,
3~ C3-Clo cycloalkyl, aryl or heterocyclic group,
wherein the substituent is selected from F, Cl, Br,
N02, R100-, Rl lS(O)m-, RlOC(O)NR10-,
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(R1o)2Nc(o)-~ CN, (R1o)2N-c(NRlo)-~ R10C(o)-,
CON(R10)2-, N3, -N(R10)2, Rl lOC(O)NR10- and
Cl-C20 alkyl, and
b) C 1 -C6 alkyl .substituted with an unsubstituted or
substituted group selected from aryl, heterocycle and
C3-Clo cycloalkyl;
R5b is .selected from:
a) hydrogen, and
b) Cl-C3 alkyl;
R6 is independently selected from hydrogen or C 1 -C6 alkyl;
R~ is independently selected from:
a) hydrogen,
b) Cl-C6 alkyl, C2-c6 alkenyl, C2-c6 alkynyl, Cl-c6
perfluoroalkyl, F, Cl, R100-, RlOC(O)NR10-, CN, N02,
(R10)2N-C(NR10)-, R10C(o)-, CON(R10)2-, -N(R10)2, or
R 1 1 OC(O)NR 10, and
c) C l -C6 alkyl substituted by C l -C6 perfluoroalkyl, R 1 0O-,
RlOC(o)NRlo-~ (R10)2N-C(NR10)-, RlOC(O)-.
CON(R 1~)2-, -N(R 1~)2, or R 1 1 OC(O)NR 10;
R9 is selected from:
a) hydrogen,
b) C2-C6 alkenyl, C2-C6 alkynyl, Cl-C6 perfluoroalkyl,
F, Cl, R10O-, R1 lS(O)m-, R10C(O)NRl0-~ CN, NO2,
(R 1 0)2N-C(NR 10), R I ~C(O)-, CON(R 1~)2-, -N(R 1~)2, or
R l l OC(O)NR 1 0-, and
c) Cl -C6 alkyl unsubstituted or substituted by Cl -C6
perfluoroalkyl, F, Cl, R 1 0O-, R 1 I S(O)m-, R 1 0C(O)NR 1 0-,
CN, (R10)2N-C(NR10)-, R10C(o)-, CON(R10)2-,
-N(R10)2, or Rl lOC(O)NR10-;
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R10 is independently selected from hydrogen, Cl-C6 alkyl, benzyl and
aryl;
R11 is independently selected from Cl-C6 alkyl and aryl;
Q is selected from:
N~ and -~- N~
~0 AI and A2areindependentlyselectedfrom: abond,-CH=CH-,-C-C-,
-C(O)-, -C(O)NR10-, O, -N(R10)-, or S(O)m;
V is selected from:
a) hydrogen,
b) heterocycle selected from pyrrolidinyl, imidazolyl, pyridinyl,
thiazolyl, pyridonyl, 2-oxopiperidinyl, indolyl, quinolinyl,
isoquinolinyl, and thienyl,
c) aryl,
d) Cl-C20 alkyl wherein from 0 to 4 carbon atoms are replaced
with a heteroatom selected from O, S, and N, and
e) C2-C20 alkenyl, and
provided that V is not hydrogen if A 1 is S(O)m and V is not hydrogen if
A 1 is a bond, n is 0 and A2 is S(O)m;
25 W is a heterocycle selected from pyrrolidinyl, imidazolyl, pyridinyl,
thiazolyl, pyridonyl, 2-oxopiperidinyl, indolyl, 4uinolinyl, or
isoquinolinyl;
- X, Y and Z are independently H2 or O;
mis 0,1 or2;
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nis 0, 1,2,30r4;
pis 0, 1,2,30r4;
r is O to 5, provided that r is O when V is hydrogen;
tis 3,40r5;and
uis Oorl;
or the pharmaceutically acceptable salts, hydrates, crystal forms and
isomers thereof.
In an even more preferred embodiment of this invention, the
Ras farnesyl transferase inhibitors are illustrated by the Formula II:
V Al(CRla2)nA2(CR1a2),.-(W)- (CR1b2)p~N~N~'
R4a
wherein:
Rla is independently selected from: hydrogen or Cl-C6 alkyl;
Rlb is independently selected from:
a) hydrogen,
b) aryl, heterocycle, cycloalkyl, R 10o-, -N(R 1 ~)2 or alkenyl,
c) Cl-C6 alkyl unsubstituted or substituted by aryl,
heterocycle, cycloalkyl, alkenyl, R100-, or -N(R10)2;
R2 or R3 are combined with R6 to form a ring such that
~;N~, iS ~H2~t;
R2 R3 R7a R7b
R4a is independently selected from:
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- 14-
a) hydrogen,
b) C 1 -C6 alkyl unsubstituted or sub.stituted by alkenyl, R 1 0O-,
Rl lS(O)m-, R10C(o)NRl0-, CN, N3, (R10)2N-C(NR10)-,
R 10c(O)-, CON(R 1~)2-, -N(R10)2, or R 1 lOC(O)NR 10,
c) aryl, heterocycle, cycloalkyl, alkenyl, R10O-,
Rl lS(O)m-, R10C(O)NR10-, CN, NO2,
(R 10)2N C(NR10), R 10c(O)-, CON(Rl0)2-~ N3,
-N(R 1 0)2, or R1 1 OC(O)NR 1 0-, and
d) Cl-C6 alkyl substituted with an unsubstituted or
substituted group selected from aryl, heterocyclic and
C3-C1o cycloalkyl;
R4b and R7b are hydrogen;
RSa is selected from:
a) substituted or unsubstituted Cl-C1o alkyl, C2-Clo alkenyl,
C3-Clo cycloalkyl, aryl or heterocyclic group,
wherein the substituent is selected from F, Cl, Br,
NO2, R 1 0O, R 1 1 S(O)m-, R 1 0C(O)NR 1 0-,
(R10)2Nc(o)-~ CN, (R10)2N-C(NR10)-, R10C(O)-,
CON(R 1~)2-, N3, -N(R 1~)2, R 1 1 OC(O)NR 10 and
Cl-C20 alkyl, and
b) C 1 -C6 alkyl substituted with an unsubstituted or
substituted group selected from aryl, heterocycle and
C3-Clo cycloalkyl;
RSb i~s selected from:
a) hydrogen, and
b) C1-C3 alkyl;
R6 is independently selected from hydrogen or C1-C6 alkyl;
- R8 is independently selected from:
a) hydrogen,
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WO97/36891 PCT~US9710~707
b) Cl-C6 alkyl, C2-c6 alkenyl, C2-c6 alkynyl, Cl-C6
perfluoroalkyl, F, Cl, R10O-, R10C(o)NR10-, CN, NO2,
(R10)2N-C(NR10)-, R10C(o)-, CON(R10)2-, -N(R10)2, or
~ R 1 1 OC(O)NR 10, and
c) Cl-C6 alkyl substituted by Cl-C6 perfluoroalkyl, R10O-,
RlOC(o)NRlo-~ (R10)2N-C(NR10)-, RlOC(O)-,
CON(R10)2-, -N(R10)2, or Rl lOC(O)NR10-;
R9 is .selected from:
a) hydrogen,
b) C2-C6 alkenyl, C2-C6 alkynyl, Cl-C6 perfluoroalkyl,
F, Cl, R10O-, Rl lS(O)m-, R10C(O)NRl0-~ CN, NO2,
(R10)2N-C(NR10)-, R10C(o)-, CON(R10)2-, -N(R10)2, or
R 1 1 OC(O)NR 10, and
c) Cl-C6 alkyl unsubstituted or substituted by Cl-C6
perfluoroalkyl,~F, Cl, R 1 0O-, R 1 1 S(O)m-~ R 1 0C(O)NR 10,
CN, (R10)2N-C(NR10)-, R10C(o)-, CON(R10)2-,
-N(R10)2, or Rl lOC(O)NR10-;
20 R10 is independently selected from hydrogen, Cl-C6 alkyl, benzyl and
aryl;
R l 1 is independently selected from Cl-C6 alkyl and aryl;
25 Q is selected from:
~r
N~ and -~- N~
A l and A2 are independently selected from: a bond, -CH=CH-~ -C_C-,
-C(O)-, -C(O)NR 10, o, -N(R 10) , or S(O)m;
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V is selected from:
a) hydrogen,
b) heterocycle ~selected from pyrrolidinyl, imidazolyl, pyridinyl,
thiazolyl, pyridonyl, 2-oxopiperidinyl, indolyl, 4uinolinyl,
isoquinolinyl, and thienyl,
c) aryl,
d) C1-C20 alkyl wherein from O to 4 carbon atoms are replaced
with a heteroatom selected from 0, S, and N, and
e) C2-C20 alkenyl, and
10 provided that V is not hydrogen if A 1 is S(O)m and V is not hydrogen if
Al is a bond, n i,s O and A2 is S(O)m;
W is a heterocycle selected from pyrrolidinyl, imidazolyl, pyridinyl,
thiazolyl, pyridonyl, 2-oxopiperidinyl, indolyl, quinolinyl, or
1 S isoquinolinyl;
X, Y and Z are independently H2 or 0;
m is 0, 1 or 2;
nis 0,1,2,30r4;
pis 0, 1,2,30r4;
r is O to 5, provided that r is O when V is hydrogen;
tis 3,40r5;and
uis Oorl;
25 or the pharmaceutically acceptable salts, hydrates, crystal forms or
isomers thereof.
In yet a more preferred embodiment of this invention, the
Ras farnesyl transferase inhibitors are illustrated by the Formula rv:
CA 022496l7 l998-09-22
W 097/36891 PCT~US97tO5707
V A1(CR1a2)nA2(C~1a2)n-(W)- (CR1b2)p~l~N~ R
lV O R4b
R a
wherein:
5 Rla is independently selected from: hydrogen or C1-C6 alkyl;
R 1 b is independently selected from:
a) hydrogen,
b) aryl, heterocycle, cycloalkyl, R100-, -N(R 1~)2 or alkenyl,
c) C1-C6 alkyl unsubstituted or substituted by aryl,
heterocycle, cycloalkyl, alkenyl, R10O-, or-N(R10)2;
R4a and R7a are independently selected from:
a) hydrogen,
b) C1 -C6 alkyl unsubstituted or substituted by alkenyl, R 10o-,
Rl lS(O)m-, R10C(O)NRl0-~ CN, N3, (R10)2N-C(NR10)-,
R 1 ~C(O)-, CON(R 1~)2-, -N(R 1~)2, or R I 1 OC(O)NR 10 ,
c) aryl, heterocycle, cycloalkyl, alkenyl, R10O-,
R 1 1S(O)m-, R10C(o)NR10-, CN, NO2, (R10)2N-
C(NR 10), R 10c(O)-, CON(R 1~)2-, N3,
-N(R10)2, or R1 1OC(O)NR10-, and
d) C 1 -C6 alkyl substituted with an unsubstituted or
substituted group selected from aryl, heterocyclic and
C3-Clo cycloalkyl;
- R4b is hydrogen;
RSa is ~selected from:
a) substituted or unsub~stituted Cl-C1o alkyl, C2-Clo alkenyl,
C3-Cl o cycloalkyl, aryl or heterocyclic group,
CA 02249617 1998-09-22
W O97136891 PCTAUS97/05707
wherein the substituent is selected from F, Cl, Br,
NO2, R l Oo, R I 1 S(O)m-~ R I OC~O)NR 10,
(R 1 0)2NC(o)-, CN, (R 1 0)2N-C(NR 10) , R 1 ~C(O)-,
CON(R10)2-, N3, -N(R10)~, Rl lOC(O)NR10- and
Cl-C20 alkyl, and
b) C l-C6 alkyl substituted with an unsubstituted or
substituted group selected from aryl, heterocycle and
C3-Clo cycloalkyl;
~0 Rsb is selected from:
a) hydrogen, and
b) C 1 -C3 alkyl;
R8 is independently selected from:
a) hydrogen,
b) Cl-C6 alkyl, C2-c6 alkenyl, C2-C6 alkynyl, Cl-c6
perfluoroalkyl, F, Cl, R100-, R10C(O)NR10-,CN,NO2,
(Rl0)2N-c(NRlo)~Rloc(o)-~coN(Rlo)2-~-N(Rlo)2~ or
RllOC(O)NR10-, and
c) C1 -C6 alkyl substituted by Cl -C6 perfluoroalkyl, Rl Oo,
Rl0c(o)NRlo-~(Rlo)2N-c(NRlo)-~Rloc(o)
CON(R10)2-, -N(R10)2, or Rl lOC(O)NR10-;
R9is selected from:
a) hydrogen,
b) C2-C6 alkenyl, C2-C6 alkynyl, Cl-C6 pe~luoroalkyl,
F,CI,R100-,RllS(O)m-,R10C(O)NR10-,CN,NO2,
(Rl0)2N-c(NRlo)-~Rloc(o)-~coN(Rlo)2-~-N(Rlo)2~ or
RllOC(O)NR10-, and
c) C 1 -C6 alkyl unsub~tituted or substituted by C 1 -C6
perfluoroalkyl, F, Cl, R1OO-, RllS(O)m-,Rl0c(o)NR
CN,(Rl0)2N-c(NRlo)-~Rloc(o)-~coN(Rlo)2
-N(R10)2, or Rl lOC(O)NR10-;
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- 19-
R10 i.s independently selected from hydrogen, Cl-C6 alkyl, benzyl and
aryl;
~ 11 is independently selected from Cl -C6 alkyl and aryl;
Q is selected from:
N~ and -~- N _ ~
~0 Al and A2 areindependentlyselectedfrom: abond, -CH=CH-, -C_C-,
-C(O)-, -C(O)NR10-, O, -N(R10)-, or S(O)m;
V is selected from:
a) hydrogen,
lS b) heterocycle selected from pyrrolidinyl, imidazolyl, pyridinyl,thiazolyl, pyridonyl, 2-oxopiperidinyl, indolyl, quinolinyl,
isoquinolinyl, and thienyl,
c) aryl,
d) C l -C20 alkyl wherein from 0 to 4 carbon atoms are replaced
with a heteroatom selected from O, S, and N, and
e) C2-C20 alkenyl, and
provided that V is not hydrogen if Al is S(O)m and V is not hydrogen if
Al is a bond, n is 0 and A2 is S(O)m;
2;S W is a heterocycle selected from pyrrolidinyl, imidazolyl, pyridinyl,
thiazolyl, pyridonyl, 2-oxopiperidinyl, indolyl, quinolinyl, or
isoquinolinyl;
- Z is independently H2 or O;
m is 0, 1 or 2;
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- 20 -
nis 0,1,2,30r4;
pi~ 0, 1,2,30r4;
r is O to 5, provided that r is O when V is hydrogen;
tis 3,40r5; and
uis Oor l;
or the pharmaceutically acceptable salts, hydrates, crystal forms, or
isomers thereof.
In the most preferred embodiment of this invention. the Ras
farnesyl transferase inhibitors are illustrated by the Formula V:
V A (CR 2)nA (CR1a2)n -(W)- (CR1b2)p~fN~--N/~ 5b
IV O
wherein:
15 Rla is independently selected from: hydrogen or Cl-C6 alkyl;
R1b is independently selected from:
a) hydrogen,
b) aryl, heterocycle, cycloalkyl, R100-, -N(R10)2 or alkenyl,
c) Cl-C6 alkyl un~substituted or substituted by aryl,
heterocycle, cycloalkyl, alkenyl, R100-, or -N(R10)2;
R4a and R7a are independently .selected from:
a) hydrogen,
b) C l -C6 alkyl unsubstituted or substituted by alkenyl, R l Oo,
Rl lS(O)m-, RlOC(O)NRlO-, CN, N3, (R10)2N-C(NR10)-,
R 1 ~C(O)-, CON(R 1~)2-, -N(R 1~)2, or R 1 1 OC(O)NR 10,
c) aryl, heterocycle, cycloalkyl, alkenyl, R100-,
R 1 l S(O)m, R l OC(O)NR 1 0-, CN, N02,
(R10)2N-C(NR10)-, RlOC(O)-, CON(R10)2-,
N3, -N(R 1~)2, or Rl lOC(O)NR10-, and
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d) C I -C6 alkyNsubstituted with an un,substituted or
substituted group ,selected from aryl, heterocyclic and
C3-Clo cycloalkyl;
5 R5a is selected from:
a) substituted or unsubstituted Cl-C1o alkyl, C2-Clo alkenyl,
C3-Clo cycloalkyl, aryl or heterocyclic group,
wherein the substituent is selected from F, Cl, Br,
NO2, R10O-, Rl lS(O)m-, R10C(o)NR10-,
(R 1 0)2NC(O)-, CN, (R 1 0)2N-C(NR 10) , R 1 ~C(O)-,
CON(R 1~)2-, N3, -N(R 1~)2, R 1 1 OC(O)NR 10 and
C1-c2o alkyl, and
b) Cl -C6 alkyl substituted with an unsubstituted or
substituted group selected from aryl, heterocycle and
C3-C1o cycloalkyl;
R5b is selected from:
a) hydrogen, and
b) C 1 -C3 alkyl;
R~ is independently selected from:
a) hydrogen,
b) C 1 -C6 alkyl, C2-c6 alkenyl, C2-c6 alkynyl, C 1 -C6
perfluoroalkyl, F, Cl, R10O-, R10C(O)NRl0-~ CN, NO2,
(R 1 0)2N-C(NR 10)-, R 1 ~C(O)-, CON(R 1~)2-, -N(R 1~)2, or
R 1 1 OC(O)NR 10-, and
c) C I -C6 alkyl substituted by C 1 -C6 perfluoroalkyl, R 1 0O-,
RlOC(O)NR10-, (R10)2N-C(NR10)-, RlOC(O)-,
CON(R 1~)2-, -N(R 1~)2, or R 1 1 OC(O)NR 10;
R9 is selected from:
a) hydrogen,
b) C2-C6 alkenyl, C2-C6 alkynyl, C 1 -C6 perfluoroalkyl,
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W O97/36891 PCTrUS97/05707
F~ Cl, R100-, R~ lS(O)m-, RlOC(O)NR10-, CN, N02,
(R 1 0)2N-C(NR 10), R I ~C(O)-, CON(R 1~)2-, -N(R 1~)2, or
R 1 1 OC(O)NR 10, and
c) Cl-C6 alkyl unsubstituted or substituted by Cl-C6
S perfluoroalkyl, F, Cl, R 1 00-, R 1 l S(O)m-, R l OC(O)NR 10,
CN, (R 10)2N-C(NR 10), R 10C(o)-, CON(R10)2-,
-N(R10)2, or Rl lOC(O)NR10-;
R10 is independently selected from hydrogen, Cl-C6 alkyl, benzyl and
aryl;
R 1 1 is independently selected from Cl -C6 alkyl and aryl;
A 1 and A2 are independently selected from: a bond, -CH=CH-, -C_C-,
-C(O)-, -C(O)NR 10, O, -N(R10)-, or S(O)m;
V is selected from:
a) hydrogen,
b) heterocycle selected from pyrrolidinyl, imidazolyl, pyridinyl,
thiazolyl, pyridonyl, 2-oxopiperidinyl, indolyl, quinolinyl,
isoquinolinyl, and thienyl,
c) aryl,
d) Cl-C20 alkyl wherein from O to 4 carbon atoms are replaced
with a heteroatom selected from 0, S, and N, and
e) C2-C20alkenyl~ and
provided that V is not hydrogen if A 1 is S(O)m and V is not hydrogen if
A1 is a bond, n is O and A2 i.s S(O)m;
W is a heterocycle selected from pyrrolidinyl, imidazolyl, pyridinyl,
30 thiazolyl, pyridonyl, 2-oxopiperidinyl, indolyl, quinolinyl, or
isoquinolinyl;
m is 0, 1 or 2;
nis 0, 1,2,30r4;
pis 0,1,2,30r4;
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- 23 -
r is O to 5, provided that r is O when V is hydrogen:
tis 3,40r5; and
uis Oor l;
or the pharmaceutically acceptable .salt.s, hydrates, crystal form~s or
5 isomers thereof.
Examples of the compounds of this invention are as follows:
N-[ 1-[1 -(4-Cyanobenzyl)- 1 H-imidazol-5-ylacetyl]pyrrolidin-2(S)-
ylmethyl]-3(S)-ethyl-proline N-(3-chlorophenylmethyl)-amide
N- [ 1 - [ 1 -(4-Cyanobenzyl)- 1 H-imidazol-S -ylacetyl]pyrrolidin-2(S)-
ylmethyl]-3(S)-ethyl-proline N-methyl-N-(3-chlorophenylmethyl)-amide
N-Ll-l 1-(4-Cyanobenzyl)-lH-imidazol-5-ylacetyl]-3(S)-ethylpyrrolidin-
15 2(S)-ylmethyl]-3(S)-ethyl-proline N-(3-chlorophenylmethyl)-amide
N-[l-[l -(4-Cyanobenzyl)-lH-imidazol-5-ylacetyl]-3(S)-ethylpyrrolidin-
2(S)-ylmethyl]-3(S)-ethyl-proline N-methyl-N-(3-chlorophenylmethyl)-
amide
N-[1-(3-l IH-Imidazol-4-yl]propionyl)-pyrrolidin-2(S)-ylmethyl]-3(S)-
ethyl-proline-N-methyl-N-(3-chlorophenylmethyl) amide
N-[ 1-(3-[1 H-Imidazol-4-yl]propionyl)-3(S)-ethylpyrrolidin-2(S)-
25 ylmethyl]-3(S)-ethyl-proline-N-methyl-N-(3-chlorophenylmethyl) amide
N-[ 1-( I -( I -Farnesyl)- 1 H-imidazol-5-ylacetyl)-pyrrolidin-2(S)-ylrnethyl] -3(S)-ethyl-proline-N-methyl-N-(3-chlorophenylmethyl) amide
30 N-[ 1-(1-( I -Geranyl)- I H-imidazol-5-ylacetyl)-pyrrolidin-2(S)-ylmethyl] -
3(S)-ethyl-proline-N-methyl-N-(3-chlorophenylmethyl) amide
- N-[1-[1-(4-Methoxybenzyl)-lH-imidazol-5-ylacetyl]pyrrolidin-~(S)-
ylmethyl]-3(S)-ethyl-proline-N-(3-chlorophenylmethyl) amide
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- 24 -
N-[ 1-[1 -(4-Methoxybenzyl)- 1 H-imidazol-5-ylacetyl]pyrrolidin-2(S)-
ylmethyl]-3(S)-ethyl-proline-N-methyl-N-(3-chlorophenylmethyl) amide
N-[ 1-~ I -(2-Naphthylmethyl)- 1 H-imidazol-5-ylacetyl]pyrrolidin-2(S)-
ylmethyl3-3(S)-ethyl-proline-N-(3-chlorophenylmethyl) amide
N-[ 1-[1 -(2-Naphthylmethyl)- 1 H-imidazol-5-ylacetyl]pyrrolidin-2(S)-
ylmethyll-3(S)-ethyl-proline-N-methyl-N-(3-chlorophenylmethyl) amide
10 or the pharmaceutically acceptable salts thereof.
Specific examples of compound,s of the invention are:
N-[ 1-(3-[1 H-Imidazol-4-yl]propionyl)-pyrrolidin-2(S)-ylmethyl]-3(S)-
ethyl-proline-N-methyl-N-(3-chlorophenylmethyl) amide
Cl
0 ~ 9--N~
N--N~
N-[l -[1-(4-Cyanobenzyl)-lH-imidazol-5-ylacetyl]pyrrolidin-2(S)-
ylmethyl]-3(S)-ethyl-proline N-(3-chlorophenylmethyl)-amide
NC
~~
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N-[ I -L 1 -(4-Cyanobenzyl)- 1 H-imidazol-~-ylacetyl]pyrrolidin-2(S)-
ylmethyl]-3(S)-ethyl-proline N-methyl-N-(3-chlorophenylmethyl)-amide
- NC
~r~
or the pharmaceutically acceptable salts thereof.
In the present invention, the amino acids which are disclosed
are identified both by conventional 3 letter and single letter abbreviations
as indicated below:
Alanine Ala A
Arginine Arg R
Asparagine Asn N
Aspartic acid Asp D
Asparagine or
Asparticacid Asx B
Cysteine Cys C
Glutamine Gln Q
Glutamic acid Glu E
Glutamine or
Glutamic acid Glx Z
Glycine Gly G
Histidine His H
Isoleucine Ile
~5 Leucine Leu L
Lysine Lys K
Methionine Met M
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- 26 -
Phenylalanine Phe F
Proline Pro P
Serine Ser S
Threonine Thr T
Tryptophan Trp W
Tyrosine Tyr Y
Valine Val V
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.
As used herein, "alkyl" is intended to include both branched
15 and straight-chain saturated aliphatic hydrocarbon groups having the
specified number of carbon atoms.
As used herein, "cycloalkyl" is intended to include non-
aromatic cyclic hydrocarbon groups having the specified number of
carbon atoms. Examples of cycloalkyl groups include cyclopropyl,
20 cyclobutyl, cyclopentyl, cyclohexyl and the like.
"Alkenyl" groups include those groups having the specified
number of carbon atoms and having one or several double bonds.
Examples of alkenyl groups include vinyl, allyl, isopropenyl, pentenyl,
hexenyl, heptenyl, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclo-
25 hexenyl, l-propenyl, 2-butenyl, 2-methyl-2-butenyl, isoprenyl, farnesyl,
geranyl, geranylgeranyl and the like.
As used herein, "aryl" is intended to include any stable
monocyclic, bicyclic or tricyclic carbon ring(s) of up to 7 members in
each ring, wherein at least one ring is aromatic. Examples of aryl groups
30 include phenyl, naphthyl, anthracenyl, biphenyl, tetrahydronaphthyl,
indanyl, phenanthrenyl and the like.
The term heterocycle or heterocyclic, as used herein,
represents a stable 5- to 7-membered monocyclic or stable ~- to 11-
membered bicyclic or stable 1 1-15 membered tricyclic heterocycle ring
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- 27 -
which is either saturated or unsaturated, and which consists of carbon
atom~s and from one to four heteroatoms selected from the group
consisting of N, O, and S, and including any bicyclic group in which
any of the above-defined heterocyclic rings is fu~sed to a benzene ring.
5 The heterocyclic ring may be attached at any heteroatom or carbon
atom which results in the creation of a stable structure. Examples of
such heterocyclic elements include, but are not limited to, azepinyl,
benzimidazolyl, benzisoxazolyl, benzofurazanyl, benzopyranyl,
benzothiopyranyl, benzofuryl, benzothiazolyl, benzothienyl,
10 benzoxazolyl, chromanyl, cinnolinyl, dihydrobenzofuryl, dihydro-
benzothienyl, dihydrobenzothiopyranyl, dihydrobenzothio-pyranyl
sulfone, furyl, imidazolidinyl, imidazolinyl, irnidazolyl, indolinyl,
indolyl, isochromanyl, isoindolinyl, isoquinolinyl, isothiazolidinyl,
isothiazolyl, isothiazolidinyl, morpholinyl, naphthyridinyl, oxadiazolyl,
15 2-oxoazepinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl,
piperidyl, piperazinyl, pyridyl, pyridyl N-oxide, pyridonyl, pyrazinyl,
pyrazolidinyl, pyrazolyl, pyrimidinyl, pyrrolidinyl, pyrrolyl, quinazolinyl,
quinolinyl, quinolinyl N-oxide, quinoxalinyl, tetrahydrofuryl, tetrahydro-
isoquinolinyl, tetrahydro-quinolinyl, thiamorpholinyl, thiamorpholinyl
20 sulfoxide, thiazolyl, thiazolinyl, thienofuryl, thienothienyl, and thienyl.
As used herein, the terms "substituted aryl", "substituted
heterocycle" and "substituted cycloalkyl" are intended to include the
cyclic group which is .substituted with 1 or 2 substitutents selected from
the group which includes but is not limited to F, Cl, Br, CF3, NH2
25 N(CI-C6 alkyl)2, NO2, CN, (Cl-C6 alkyl)O-, -OH, (Cl-C6 alkyl)
S(O)m-, (C1-C6 alkyl)C(O)NH-, H2N-C(NH)-, (Cl-C6 alkyl)C(O)-,
(Cl-C6 alkyl)OC(O)-, N3,(CI-C6 alkyl)OC(O)NH- and Cl-C20 alkyl.
The following structure:
,N~
((~H2)t
J
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- 2~ -
represents a cyclic amine moiety having S or 6 members in the ring, such
a cyclic amine which may be optionally fused to a phenyl or cyclohexyl
ring. Examples of such a cyclic amine moiety include, but are not limited
to, the following specific structures:
5 (~ ~ ~
It is also understood that substitution on the cyclic amine moiety by R~a
and R8b may be on different carbon atoms or on the same carbon atom.
When R3 and R4 are combined to forrn - (CH2)S -, cyclic
moieties are formed. Examples of such cyclic moieties include, but are
10 not limited to:
As used herein, the phrase "nitrogen containing C4-C9
mono or bicyclic ring system wherein the non-nitrogen containing ring
may be a C5-C7 saturated ring" which defines moiety "Q" of the instant
15 invention includes but is not limited to the following ring sy.stems:
CA 02249617 1998-09-22
W 097136891 PCTrUS9~/05707
~5- N~ N~
~_N~ -~--N~,
The pharmaceutically acceptable salts of the compounds of
this invention include the conventional non-toxic salts of the compounds
S 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,
10 malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenyl-
acetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxy-benzoic,
fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic,
i~sethionic, trifluoroacetic and the like.
It is intended that the definition of any substituent or variable
l~S (e.g., R10, Z, n, etc.) at a particular location in a molecule be independent
of its definitions elsewhere in that molecule. Thus, -N(R 1~)2 represents
-NHH, -NHCH3, -NHC2Hs, etc. It is understood that substituents and
substitution patterns on the compounds of the instant invention can be
selected by one of ordinary skill in the art to provide compounds that are
20 chemically stable and that can be readily synthesized by techniques
known in the art a~s well as those methodl~i set forth below.
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- 30 -
Preferably, Rla and Rlb are independently selected from:
hydrogen, -N(R~)2, R8C(O)NR8- or Cl-C6 alkyl unsubstituted or
substi~ted by -N(R~)2, R~sO- or R8C(O)NRg-.
Preferably, R2 is the sidechain of glycine (hydrogen).
Preferably, R3 is selected from:
a) a side chain of a naturally occurring amino acid,
b) substituted or unsubstitllted Cl -C20 alkyl,
wherein the substituent is selected from F, Cl, Br,
N(R 1 0)2, NO2, R 1 0O-, R 1 1 S (~)m-, R l 0C(o)NR 10,
CN, (R 1 0~2N-C(NR 10) , R 1 ~C(O)-, CON(R 1~)2-,
N3, -N(R10)2, R1 1OC(O)NR10- and C1-C20 alkyl,
and
c) C1-C6 alkyl substituted with an unsubstituted or
substituted group selected from aryl, heterocycle
and C3-C1o cycloalkyl; or
R3 is combined with R6 to form pyrrolidinyl ring.
Preferably, R4a, R4b, R7a and R7b are independently
selected from: hydrogen, C1-C6 alkyl, aryl and benzyl.
Preferably, R5a and R5b are independently selected from:
hydrogen, unsubstituted or substituted C1-C6 alkyl, aryl, or Cl-3 alkyl
substituted with an unsubstituted or substituted group selected from aryl
or heterocycle .
Preferably, R6 is: hydrogen or is combined with R3 to form
pyrrolidinyl ring.
Preferably, R~ is selected from: hydrogen, perfluoroalkyl,
F, Cl, Br, R10O-, Rl 1S(O)m-, CN, NO2, R102N-C(NR10)-, Rl0C(o)-,
CON(R10)2-, N3, -N(R10)2, or RllOC(O)NR10- and C1-C6 alkyl.
Preferably, R9 is hydrogen.
Preferably, R 10 is selected from H, C 1 -C6 alkyl and benzyl.
Preferably, R 12 is selected from C1 -C6 alkyl and benzyl.
Preferably, Al and A2 are independently selected from:
a bond, -C(O)NR 10, -NR l ~C(O)-, O, -N(R 10) , -S(O)2N(R 10) and
-N(R I ~)S(O)2-.
Preferably, Q is a pyrrolidinyl ring.
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Preferably, V i.s selected from hydrogen, heterocycle and
aryl.
Preferably, n, p and r are independently 0, 1, or 2.
Preferably t is 3.
The pharmaceutically acceptable salts of the compounds of
this invention can be synthe.sized from the compounds of this invention
which contain a basic moiety conventional chemical methods. Generally,
the salts are prepared by ion exchange chromatography or by reacting the
free base with stoichiometric amounts or with an excess of the desired
10 salt-forming inorganic or organic acid in a suitable solvent or various
combinations of solvents.
The compounds of the invention can be synthesized
from their constituent amino acids by conventional peptide synthesis
techniques, and the additional methods described below. Standard
15 methods of peptide synthesis are di~sclosed, for example, in the following
works: Schroeder et al., "The Peptides", Vol. I, Academic Press 1965, or
Bodanszky et al., "Peptide Synthesi~", Interscience Publishers, 1966, or
McOmie (ed.) "Protective Groups in O~ganic Chemistry", Plenum Press,
1973, or Barany et al., "The Peptides: Analysis, Synthesis, Biology" 2,
20 Chapter 1, Academic Press, 1980, or Stewart et al., "Solid Phase Peptide
Synthesis", Second Edition, Pierce ChemicaJ Company, 1984. The
teachings of these works are hereby incorporated by reference.
Abbreviations used in the description of the chemistry and in
the Examples that follow are:
Ac2O Acetic anhydride;
Boc t-Butoxycarbonyl;
DBU 1,~-diazabicyclo[5.4.0]undec-7-ene;
DMAP 4-Dimethylaminopyridine;
DME 1,2-Dimethoxyethane;
DMF Dimethylformamide;
EDC 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide-
hydrochloride;
HOBT I -Hydroxybenzotriazole hydrate;
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Et3N Triethylamine;
EtOAc Ethyl acetate;
FAB Fast atom bombardment;
HOOBT 3-Hydroxy- 1 ,2,2-benzotriazin-4(3~)-one;
5 HPLC High-performance li4uid chromatography;
MCPBA m-Chloroperoxybenzoic acid;
MsCI Methane~sulfonyl chloride;
NaHMDS Sodium bis(trimethylsilyl)amide;
Py Pyridine;
TFA Trifluoroacetic acid;
THF Tetrahydrofuran.
Compounds of this invention are prepared by employing
the reactions shown in the following Reaction Schemes A-J, 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. Some key bond-forming and peptide
modifying reactions are:
Reaction A Amide bond formation and protecting group cleavage using
standard solution or solid phase methodologies.
Reaction B Preparation of a reduced peptide subunit by reductive
alkylation of an amine by an aldehyde using sodium
cyanoborohydride or other reducing agents.
Reaction C Deprotection of the reduced peptide subunit
Reaction D Amide bond formation and protecting group cleavage using
standard solution or solid phase methodologies.
~ Reaction E Preparation of a reduced subunit by borane reduction of the
amide moiety.
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Reaction Schemes A-E illustrate bond-forming and peptide
modifying reactions incorporating acyclic peptide units. It is well under-
stood that such reaction,s are equally useful when the - NHC(RA)- moiety
of the reagents and compounds illustrated is replaced with the following
5 moiety:
(C~ H2)t
~R7b
R7a
These reactions may be employed in a linear sequence to provide the
compounds of the invention or they may be used to synthesize fragments
which are ,sub,sequently joined by the reactions described in the Reaction
10 Schemes.
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- 34 -
REACTION SCHEME A
Reaction A. Couplin~ of re~sidues to form an amide bond
N I OH + ~3
~ R4b
R4a
EDC, HOBT H ~ CO2R
or HOOBT >~ R4b
2 J~ ~
R ~ Q
R4a~ R4b
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REACTION SCHEME B
Reaction B. Preparation of reduced peptide ~ubunit.s by reductive
alkylation
O RA CO2R
~ ~ R4b
NaCNBH3 ~,~0~ N~zR
R4b
/ - -
R4a
REACTION SCHEME C
Reaction C. Deprotection of reduced peptide subunits
H CO2R
>~O~N < TFA or
O RA ~ Q \
~ R4b HCI
R4a
H2N~ CO2R
- ~
RA ( Q~)_
~C,,/ R4b
R4a
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REACTION SCHEME D
Reaction D. Couplin~ of residues to form an amide bond
EDC, HOBT
~oJ~ HN - R6b Et N DMF
R4b
R4a
O ~ R5a HCI orTFA
~OJJ~ N R5b
Q \
~R4b
R4a
o
H~ ~~ ~RR55ba
~Q ~
~R4b
R4a
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REACTION SCHEME E
Reaction E. Preparation of reduced dipeptides from peptides
~, ~ < BH3 THF
O RA ~Q ~
~R4b
R4a
H CO2R
>I' o RA ~ Q
)~R4b
R4a
where RA is R2, R3, R5a or RSb as previously defined; R4a and R4b are
as previously defined; and R is an appropriate protecting group for the
carboxylic acid.
Reaction Schemes F - M illustrate reactions wherein the
10 non-sulfhydryl-con~ining moiety at the N-terminus of the compounds of
the instant invention is attached to an acyclic peptide unit which may be
further elaborated to provide the instant compounds. It is well understood
that such reactions are eclually useful when the - NHC(RA) - moiety of
the reagents and compounds illustrated is replaced with the following
1 5 moiety:
((~ H2)t
~R7b
R7a
These reactions may be employed in a linear sequence to provide the
compounds of the invention or they may be used to synthesize fragments
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- 3~ -
which are subse4uently joined by the reaction.s described in Reaction
Schemes A - E.
The intermediates whose synthesis are illustrated in
Reaction Schemes A and C can be reductively alkylated with a variety of
aldehydes, such as I, as shown in Reaction Scheme F. The aldehydes can
be prepared by standard procedures, such as that described by O. P. Goel,
U. Krolls, M. Stier and S. Kesten in Organic Syntheses. 1988, 67, 69-75,
from the appropriate amino acid (Reaction Scheme F). The reductive
alkylation can be accomplished at pH 5-7 with a variety of reducing
10 agents, such as sodium triacetoxyborohydride or sodium cyanoboro-
hydride in a solvent such as dichloroethane, methanol or dimethylforma-
mide. The product II can be deprotected to give the final compounds III
with trifluoroacetic acid in methylene chloride. The final product III is
isolated in the salt form, for example, as a trifluoroacetate, hydrochloride
15 or acetate salt, among others. The product diamine III can further be
selectively protected to obtain IV, which can subsequently be reductively
alkylated with a second aldehyde to obtain V. Removal of the protecting
group, and conversion to cyclized products such as the dihydroimidazole
VII can be accomplished by literature procedures.
Alternatively, the protected dipeptidyl analog intermediate
can be reductively alkylated with other aldehydes such as l-trityl-4-
carboxaldehyde or l-trityl-4-imidazolylacetaldehyde, to give products
such as VIII (Reaction Scheme G). The trityl protecting group can be
removed from VIII to give IX, or alternatively, VIII can first be treated
25 with an alkyl halide then subsequently deprotected to give the alkylated
imidazole X. Alternatively, the dipeptidyl analog interrnediate can be
acylated or sulfonylated by standard techniques.
The imidazole acetic acid XI can be converted to the acetate
XIII by standard procedures, and XIII can be first reacted with an alkyl
30 halide, then treated with refluxing methanol to provide the regiospecific-
ally alkylated imidazole acetic acid ester XIV. Hydrolysis and reaction
with the protected dipeptidyl analog intermediate in the presence of
condensing reagents such as 1-(3-dimethylaminopropyl)-3-ethylcarbo-
diimide (EDC) leads to acylated products such as XV.
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If the protected dipeptidyl analog intermediate is reductively
alkylated with an aldehyde which also has a protected hydroxyl group,
such as XVI in Reaction Scheme I, the protecting groups can be
subse4uently removed to unmask the hydroxyl group (Reaction Schemes
1, J). The alcohol can be oxidized under standard conditions to e.g. an
aldehyde, which can then be reacted with a variety of organometallic
reagents such as Grignard reagents, to obtain secondary alcohols such
as XX. In addition, the fully deprotected amino alcohol XXI can be
reductively alkylated (under conditions described previously) with a
10 variety of aldehydes to obtain secondary amines, such as XXII (Reaction
Scheme K), or tertiary amines.
The Boc protected amino alcohol XVIII can also be utilized
to synthesize 2-aziridinylmethylpiperazines such as XXIII (Reaction
Scheme L). Treating XVIII with l,l'-sulfonyldiimidazole and sodium
15 hydride in a solvent such as dimethylformamide led to the forrnation of
aziridine XXIII . The aziridine reacted in the presence of a nucleophile,
such as a thiol, in the presence of base to yield the ring-opened product
XXIV .
In addition, the protected dipeptidyl analog intermediate can
20 be reacted with aldehydes derived from amino acids such as O-alkylated
tyrosines, according to standard procedures, to obtain compounds such as
XXX, as shown in Reaction Scheme M. When R' is an aryl group, XXX
can first be hydrogenated to llnm~;k the phenol, and the amine group
deprotected with acid to produce XXXI. Alternatively, the amine
25 protecting group in XXX can be removed, and O-alkylated phenolic
amines such as XXXII produced.
Similar procedures as are illustrated in Reaction Schemes
F-M may be employed using other peptidyl analog intermediates such
as those whose synthesis is illustrated in Reaction Schemes B - E.
Reaction Schemes N-R illustrate syntheses of suitably
substituted aldehydes useful in the syntheses of the instant compounds
wherein the variable W is present as a pyridyl moiety. Similar synthetic
strategies for preparing alkanols that incorporate other heterocyclic
moieties for variable W are also well known in the art.
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REACTION SCHEME F
Boc NH~
2 ~ CO2R Boc NH CHO
R ~D Q ~ NaBH(OAc)3
~ R4b Et3N, CICH2CH2CI
R a
NHBoc
~_H Y CO R CF3co2H
Boc NH N~JI~ / 2
,~f\ CH2CI2
R ~ Q ~
R4a~ R4b
NH2
NH/~ N~JI~ ~02R Boc20
RA l~ Q ~ CH2CI2
R4a>~ R4b
~ H2 y CO2R ~CHO
BocN H N~\ <
- ~ NaBH(OAc)3
Q ) Et3N, CIC
IV ~ R4b
R4a
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REACTION SCHEME F (continued)
/=\
~,
NH CF3CO2H, C H2CI2;
BocNH/~ NH~I~ CO2R NaHCO3
R ~ Q ~
~ R4a~ R4b
NH
2 ~ ~ ~NC
RA ~ Q \ AgCN
Vl ~R4b
R4a
H Y
N~l~ CO2R
/~ RA ~ Q ~
N~,N~ '~,1~ R4b
R4a
Vll
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REACTION SCHEME G
y
H2N J~ &o2R NaBH(OAc)3
Et3N, CICH2CH2CI
R
R4a~ R4b ~(CH2)nCHO
Tr
H2)n+1 ,JI, <
~C ~ R
~4a
Tr ~ ~ 1 ) Ar CH2X, CH3CN
VHI 2) CF3C02H, CH2C12
CF3CO2H, CH2CI2 (c2H5)3siH
(C2H5)3siH
H Y CO2R
(CH2)n~ ~\ ~'<
N ~ R4a~ R4b
H IX
X R
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REACTION SCHEME H
N~ 2CO2H CH N_~CH2C02CH3
H HCI NH . HCI
Xl Xll
N CH2C02CH31 ) ArCH2X CH3CN
(c6H5)3cBr ~ reflux
(C2Hs)3N N 2) CH30H, retlux
DMF Tr
Xlll
Ar--\N ~CH2c~2cH3 2.5N HClaq
~ 55~C
N
XIV
Ar~\N~C H2CO2H
N
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- 44 -
REACTION SCHEME I
H2N ~ ~ 2
R4a~ R4b
EDC HCI
HOBt
DMF
Ar~ ~<~
XV R4a R4b
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REACTION SCHEME J
NaBH(OAc)3
CO2R Et3N, CICH2CH2CI
H2N J~ (
RA ~ Q \~ BnOl
~ R4b BocNH CHO
R4a
XVI
NHBoc
/~N~JI~ CO2R 20% Pd(OH)2 H2
BnO H J~l--< CH30H
( Q ~ CH3CO2H
XV I 1 4a ~ R4b
R
NHBoc
~NJ~ CO2R CICOCOCI
HO H ,N ~ DMSO CH2C12
RA ( Q~) (C2H5)3N
\/ /--D 4b
R 4a n
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REACTION SCHEME J (CONTINUED)
H NHBoc y
O~HN~J~ < (c2H6)~o
RA ~ Q ~ 2. TFA, CH2C12
~R4b
XIX R4a/
R' NH2
HO>~-- Y CO R
RA /~Q ~
XX ~ R4b
R4a
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REACTlON SCHEME K
NHBoc y CF3CO2H
HO/~H JI, CO2R CH2CI2
RA I~Q ~
XVIII ~__~ R4b
R4a
NH2 y R'CHO
~( 11 C02R
HO \~HN ~ < NaBH(OAc)3
~) CICH2CH2CI
~/ ~ r~ 4b
R4a n
R'CH2~
NH y
HO/~H~ CO2R
RA ~Q ~
XXII ~ R4b
R4a
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- 4~ -
REACTION SCHEME L
NHBoc H H
H~2R ~N~S,N~
XVIII R4b NaH, DMF 0~C
R4a
RA Q< (C2Hs)3N
XXI I I ~ R4b C H30H
R4a
NH2 y
R'S/~H \J~ CO2R
RA ~Q ~
XXIV ~ R4b
R4a
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- 49 -
REACTION SCHEME M
HO~ 1) Boc20, K2C~3 HO,~
THF-H20
2) C H2N2, EtOAc ~
H2NCO2H BocNH CO2CH3
XXV XXVI
HO~
LiAlH4 ~1~ R'CH2X
TH F 1 Cs2CO3
0-20~C BocNH CH2OH DMF
XXVII
R'CH20,~ pyridine SO )~
DMSO
H (C2Hs)3N BocNH CHO
BocNH CH2O 20~C
XXVIII XXIX
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REACTION SCHEME M (continued)
R'CH o~3 ~
BocNH CHO R4a R4b
XXIX
NaBH(OAc)3
CICH2CH2CI
NHBoc
R'CH20~ R
XXX R4b
R4a
1) 20% Pd(OH)2 / HC~OAc
CH30H, CH3C02H
R'CH 0~( ~R4b
XXXI R4b
R4a
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REACTION SCHEME N
CH3 1) HN02,Br2 ~CO2CH3
~ 2) KMnO4 l ll
H2N N~ 3) MeOH,H+ Br~'Nf
R6
~\~\ MgCI R6
Zncl2lNicl2(ph3p)2 ~,C~2CH3
NaBH4 (excess) ~,CH20H
R6
SO3-Py, Et3N ~CHO
DMSO N
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- 52 -
REACTION SCHEME P
R6
R6 [~
~CO2CH3 ~\MgCI ~CO2CH3
Zn, CuCN
R6 R6
NaBH4 ~ SO3Py, Et3N ~
(excess) [~,CH20H DMSO ~CHO
R6 R6
Br~ ~ ~CO2CH3 ~ \ 9 ¢ ~ ~3"CO2CH3
ZnC12, NiC12(Ph3P)2 N
R6 R6
(excess) ~ SO3 Py Et3N ¢~ CHO
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REACTION SCHEME O
CO2CH3
Br~1. LDA, CO2 Br~
N2. MeOH, H+ N
R6 R6
~/\ MgCI 1~ CO2CH3
ZnCI2, Nicl2(ph3p)2
N
-
R6
NaBH4 (excess) ~OH S03 Py, Et3N
DMSO
N
R6
CHO
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REACTION SCHEME R
CO CH
1. LDA, CO2 ~ Br
2. (CH3)3SiCHN2
R6 ~ Br R6 ~
Zn, NiC12(Ph3P)2 Nl~co2cH3
excess NaBH4 ~1~ SO3Py, Et3N
N~CH20H DMSO
R6 3~
N~CHO
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REACTION SCHEME S
Reaction S. Alkylation of an amide
o
O ~ R5a
~ Q + 5bR -X NDaMHF
~R4b
R a
O ~ R5a HCI 0, TFA
'1 11 I--N
~'0'~,~~~ R
Q ~
~R4b
R4a
o
--N' R
- 5b
~ Q
~R4b
R4a
The instant compounds are useful as pharmaceutical
agents for m~mm~ls, especially for humans. These compounds may be
administered to patients for use in the treatment of cancer. Examples
10 of the type of cancer which may be treated with the compounds of this
invention include, but are not limited to, colorectal carcinoma, exocrine
pancreatic carcinoma, myeloid leukemias and neurological tumor.s. Such
tumors may ari~se by mutations in the ) as genes themselves, muta~ions in
the proteins that can regulate Ras formation (i.e., neurofibromin (NF- 1),
15 neu, scr, abl, lck, fyn) or by other mechanisms.
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The compounds of the instant invention inhibit farnesyl-
protein transferase and the farnesylation of the oncogene protein Ras.
The instant compounds may also inhibit tumor angiogenesis, thereby
affecting the growth of tumors (J. Rak et al. Cance1 Research, 55:4575-
5 45~0 (1995)). Such anti-angiogenesis 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 proliferative diseases, both benign and malignant,
lO wherein Ras proteins are aberrantly activated as a result of oncogenic
mutation in other genes (i.e., the Ras gene itself is not activated by
mutation to an oncogenic form) with said inhibition being accomplished
by the administration of an effective amount of the compounds of the
invention to a m~mm~l in need of such treatment. For example, a
15 component of NF-1 is a benign proliferative disorder.
The instant compounds may also be useful in the treatment
of certain viral infections, in particular in the treatment of hepatitis delta
and related viruses (J.S. Glenn et al. Science, 256:1331-1333 (1992).
The compounds of the instant invention are also useful in
20 the prevention of restenosis after percutaneous translllmin~l coronary
angioplasty by inhibiting neointim~l formation (C. Indolfi et al. Natu~ e
medicine, 1:541-545(1995).
The instant compounds may also be useful in the treatment
and prevention of polycystic kidney disease (D.L. Schaffner et al.
25 American Journal of Pathology, 142:1051-1060 (1993) and ,3. Cowley,
Jr. et al.FASEB Journal, 2:A3160 (1988)~.
The compounds of this invention may be ~r~ministered to
m~mm~ls, preferably humans, either alone or, preferably, in combination
with pharmaceutically acceptable carriers or diluents, optionally with
30 known adjuvants, such as alum, in a pharmaceutical composition,
according to standard pharmaceutical practice. The compounds can be
administered orally or parenterally, including the intravenous, intra-
muscular, intraperitoneal, subcutaneous, rectal and topical routes of
administration .
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For oral use of a chemotherapeutic compound according to
this invention, the selected compound may be admini.stered, for example,
in the form of tablets or capsules, or a.s an aqueous solution or .suspen-
sion. In the case of tablets for oral use, carriers which are commonly
used include lactose and corn starch, and lubricating agents, such as
magnesium .stearate, are commonly added. For oral administration in
capsule form, useful diluents include lactose and dried corn starch. When
aqueous suspensions are required for oral use, the active ingredient is
combined with emulsifying and suspending agents. If desired, certain
sweetening and/or flavoring agents may be added. For intramuscular,
intraperitoneal, subcutaneous and intravenous use, sterile solutions of
the active ingredient are usually prepared, and the pH of the solutions
should be suitably adjusted and buffered. For intravenous use, the total
concentration of solutes should be controlled in order to render the
preparation isotonic.
The present invention also encompasses a pharmaceutical
composition useful in the treatment of cancer, comprising the a-lmini-
stration of a therapeutically effective amount of the compounds of this
invention, with or without pharmaceutically acceptable carriers or
diluents. Suitable compositions of this invention include aqueous
solutions comprising compounds of this invention and pharmacologically
acceptable carriers, e.g., saline, at a pH level, e.g., 7.4. The ,solutions may
be introduced into a patient's intramuscular blood-.stream by local bolus
injection.
When a compound according to this invention is
administered into a human .subject, the daily dosage will normally be
determined by the prescribing physician with the dosage generally
varying according to the age, weight, and response of the individual
patient, as well as the severity of the patient's .symptoms.
In one exemplary application, a suitable amount of
compound is ~lministered to a m~mm~l undergoing treatment for cancer.
Administration occurs in an amount between about 0.1 mg/kg of body
weight to about 20 mgtkg of body weight per day, preferably of between
0.5 mg/kg of body weight to about 10 mg/kg of body weight per day.
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5~
The compounds of the instant invention are also useful
as a component in an a~ssay to rapidly determine the presence and
quantity of farnesyl-protein transferase (FPTa.se) in a composition.
Thus the composition to be tested may be divided and the two portions
5 contacted with mixtures which comprise a known substrate of FPTase
(for example a tetrapeptide having a cysteine at the amine terminu~)
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
10 farnesylate the substrate, the chemical content of the assay mixtures
may be determined by well known immunological, radiochemical or
chromatographic techniques. Because the compounds of the instant
invention are selective inhibitors of FPTase, absence or quantitative
reduction of the amount of substrate in the assay mixture without the
15 compound of the instant invention relative to the presence of the
unchanged substrate in the assay cont~ining the instant compound is
indicative of the presence of FPTase in the composition to be tested.
It would be readily apparent to one of ordinary skill in the
art that such an assay as described above would be useful in identifying
20 tissue samples which contain farnesyl-protein transferase and quantitating
the enzyme. Thus, potent inhibitor compounds of the instant invention
may be used in an active site titration assay to determine the quantity of
enzyme in the sample. A series of samples composed of aliquots of a
tissue extract containing an unknown amount of farnesyl-protein trans-
25 ferase, an excess amount of a known substrate of FPTase (for examplea 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
30 that has a Ki substantially smaller than the concentration of enzyme in the
assay vessel) required to inhibit the enzymatic activity of the sample by
50% is approximately equal to half of the concentration of the enzyme in
that particular sample.
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EXAMPLES
Examples provided are intended to assist in a further under-
standing of the invention. Particular materials employed, species and
5 conditions are intended to be further illustrative of the invention and not
limitative of the reasonable scope thereof.
The standard workup referred to in the examples refers to
solvent extraction and washing the organic solution with 10% citric acid,
10% sodium bicarbonate and brine as appropriate. Solutions were dried
10 over sodium sulfate and evaporated in vac~o on a rotary evaporator.
EXAMPLE 1
Preparation of N-l l -(3-[1 H-Imidazol-4-yl]propionyl)-pyrrolidin-2(S)-15 ylmethyl]-3(S)-ethyl-proline-N-methyl-N-(3-chlorophenylmethyl) amide
Step A: Diethyl l-acetyl-5-hydroxy-3-ethylpyrrolidine-2,2-
dicarboxylate
Sodium (4.02 g, 0.175 mol) was dissolved in a stirred
20 solution of diethyl acetamidomalonate (235.4 g, 1.19 mol) in abs EtOH
(1.4 L) at ambient temperature under argon. The reaction mixture was
cooled to 0~C, and trans-2-pentenal (100 g, 1.0~ mol) was added drop-
wise maintaining the reaction temperature at <5~C. After the addition,
the reaction was allowed to warm to room temperature, stirred for 4 h,
25 then quenched with acetic acid (28 mL). The solution was concentrated
in vacuo, and the residue dissolved in EtOAc (1.5 L), washed with 10%
NaHCO3 solution (2 x 300 mL), brine, and dried (MgSO4). The solution
was filtered and concentrated to 700 mL, then heated to reflux and treated
with hexane (1 L). On cooling, the title compound precipitated and was
30 collected, mp 106 - 109~C. 1 H NMR (CD30D) ~ 5.65 (d, 1 H, J= 5 Hz),
4.1 - 4.25 (m, 4H), 2.7-2.~s (m, lH), 2.21 (s, 3H), 2.10 (dd, lH, J = 6, 13,
Hz),1.~6- 1.97 (m, 2H), 1.27 (t, 3H, J= 7 Hz), 1.23 (t, 3H, J= 7 Hz), 1.1-
1.25 (m, lH)~ 0.97 (t, 3H, J= 7 Hz).
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Step B: Diethyl l -acetyl-3-ethylpyrrolidine-2,2-dicarboxylate
To a solution of diethyl l-acetyl-S-hydroxy-3-ethyl-
pyrrolidine-2,2-dicarboxylate (287 g, 0.95 mol) and triethylsilane (228
mL, 1.43 mol) in CH2C12 (3 L) under argon was added trifluoroacetic
acid (735 mL, 9.53 mol) dropwise with stirring while maintaining the
internal temperature at 25 ~C by means of an ice bath. After stirring for
3 h at 23~C, the solution was concentrated in vacuo,, the residue diluted
with CH2C12 (1.5 L), then treated with H2O (1 L) and solid Na2CO3
with vigorou.s stirring until the solution was basic. The organic layer was
~separated, dried (Na2SO4), filtered, then concentrated to give the title
compound as a yellow oil which was used without further purification.
Step C: 3-Ethylproline hydrochloride (Cis:Trans Mixture)
Diethyl 1-Acetyl-3-ethylpyrrolidine-2,2-dicarboxylate (373
g, 0.95 mol) was suspended in 6N HCI (2 L) and HOAc (500 mL) and
heated at reflux for 20 h ~e reaction mixture was cooled, washed with
EtOAc (lL), then concentrated in vacuo to give an oil which crystallized
upon trituration with ether to give the title compound. lH N~IR (D20) ~
4.23 (d, lH, J- 8 Hz), 3.84 (d, lH, J= 8 Hz), 3.15- 3.4 (m, 4H), 2.33- 2.44
(m, lH), 2.19-2.4 (m, lH), 2.02- 2.15 (m, 2H), 1.53- 1.72 (m, 3H), 1.23-
1.43 (m, 2H), 1.0- 1.15 (m, IH), 0.75 - 0.83 (m, 6H).
Step D: N-[(tert-Butyloxy)carbonyl]-c~is:trans-3-ethylproline methyl
ester
3-Ethylproline hydrochloride (Cis:Trans Mixture) (20 g,
0.11 mol) was dissolved in CH30H (200 mL), and the solution was
saturated with HCI gas, then stirred at 23~C for 24 h. Argon was bubbled
through the solution to remove excess HCl. The .solution was treated
with NaHCO3 (>84 g) to a pH of 8, then di-tert-butyl dicarbonate (25.1
g, 0.115 mol) dissolved in CH30H (20 mL) was added slowly. After
stirring for 18 h at 23~C, the mixture wa~s filtered, the filtrate concen-
trated, and the residue triturated with EtOAc, filtered again, and
concentrated to give the title compound as an oil.
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Step E: N-[(te) t-Butyloxy)carbonyl]-tran.~-3-ethylproline and N-
I (ter~-Butyloxy)carbonyll-cis-3-ethylproline methyl e~ster
N-l(tert-Butyloxy)carbonyl]-c~is,t)~ans-3-ethylproline methyl
ester (29.1 g, 0.113 mol) was dissolved in CH30H (114 mL) with cooling
5 to 0~C, then treated with 1 N NaOH (114 mL). After stirring for 20 h at
23~C, the solution was concentrated to remove the CH30H and then
extracted with EtOAc (3 x). The organic layers were combined, dried
(MgSO4), filtered, and concentrated to give 12.8 g of N-[(tert-
Butyloxy)carbonyl]-cis-3-ethylproline methyl ester as an oil. The
10 aqueous layer was acidified with solid citric acid and extracted with
EtOAc (2 x), the organic layers combined, dried (MgSO4), filtered, and
concentrated to give N-[(tert-Butyloxy)carbonyl]-trans-3-ethylproline a~s
an oil. lH NMR (CD30D) â 3.86 and 3.78 (2 d, lH, J = 6 Hz), 3.33 -
3.58 (m, 2H), 2.01 - 2.22 (m, 2H), 1.5 - 1.74 (m, 2H), 1.33 - 1.5 (m, lH),
1.45 and 1.42 (2 s, 9H), 0.98 (t, 3H, J= 8 Hz).
Step F: 3(S)-Ethyl-2(S)-proline hydrochloride
N-[(tert-Butyloxy)carbonyl]-t~ans-3-ethylproline (15.5 g,
0.064 mol), S-a-methylbenzylamine (9.03 mL, 0.070 mol), HOBT (10.73
20 g, 0.70 mol), and N-methylmorpholine (8 mL, 0.076 mol) were dissolved
in CH2C12 (150 mL) with stirring in an ice-H2O bath, treated with EDC
(13.4 g, 0.070 mol) stirred at 23~C for 48 h. The reaction mixture was
partitioned between EtOAc and 10% citric acid solution, the organic layer
washed with saturated NaHCO3 solution, brine and dried (MgSO4),
25 filtered, and concentrated to give an oil. This oil was dissolved in a
minimllm amount of ether (10 mL) to crystallize the desired S,S,S
diastereomer (4.2 g), mp 118- 121 ~C. A solution of this product in 8N
HCl (87 mL) and glacial acetic acid (22 mL) was heated at reflux
overnight. The solution was concentrated on a rotary evaporator, and the
30 residue taken up in H20 and extracted with ether. The aqueous layer was
concentrated to drynes.s to give a 1: 1 mixture of 3(S)-ethyl-2(S)-proline
hydrochloride and oc-methylbenzylamine.
3(S)-Ethyl-2(S)-proline containing oc-methylbenzylamine
(2.0 g, 0.0128 mol) was dissolved in dioxane (10 mL) and H2O (10 mL)
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with stirring and cooling to 0~C. N,N-diisopropylethylamine (2.2 mL,
0.012~S mol) and di-te~t-butyl-dicarbonate (2.79 g, 0.012~ mol) were
added and stirring was continued at 23~C for 4~ h. The reaction
mixture was partitioned between EtOAc (60 m~) and H2O (30 mL), the
organic layer washed with 0.5N NaOH (2 x 40 mL), the aqueous layer~
combined and washed with EtOAc ( 30 mL) and this layer back-extracted
with 0.5 N NaOH (30 mL). The aqueous layers were combined and care-
fully acidified at 0~C with 1 N HCI to pH 3. This mixture was extracted
with EtOAc (3 x 40 mL), the organ~cs combined, dried (MgSO4),
filtered and concentrated to give N-[(tert-Butyloxy)carbonyl-3(S)-ethyl-
2(S)-proline as a colorless oil. N-[(tert-Butyloxy)carbonyl-3(S)-ethyl-
2(S)-proline was dissolved in EtOAc (50 mL) and the solution was
saturated with HCI gas with cooling in an ice-H2O bath. The solution
was stoppered and stirred at 0~C. for 3 hr. Argon was bubbled through
the solution to remove excess HCI, and the solution wa~s concentrated to
dryne,ss to give 3(S)-ethyl-2(S)-proline hydrochloride.
Step G: N-~(t-Butyloxycarbonyl)-pyrrolidin-2(S)-ylmethyl]-3(S)-
ethyl-proline
3(S)-Ethyl-2(S)-proline hydrochloride (from Example 1,
Step F) (2.33 g, 0.013 mol) was dissolved in CH30H (20 mL), treated
with 3A molecular sieve~s (2 g) and KOAc (1.27 g, 0.013 mol) to adjust
the pH of the reaction mixture to 4.5-5, then N-[(te1 t-Butyloxy)
carbonyl-prolinal (Pettit et al., J. Org. Chem. (1994) 59, L21] 6287-95)
(3.36 g, 0.017 mol) was added, and the mixture was stirred for 16 hours
at room temperature. The reaction mixture was filtered, quenched with
aqueous saturated NaHCO3 (5 mL) and concentrated to dryness. The
residue was extracted with CHC13. The extract was dried (MgSO4),
filtered, and concentrated to give the title compound and inorganic .salt~.
Step H: N-[(t-Butyloxycarbonyl)-pyrrolidin-2(S)-ylmethyl]-3(S)-
ethyl-proline-N-(3-chlorophenylmethyl) amide
N-[(t-Butyloxycarbonyl)-pyrrolidin-2(S)-ylmethyl] -3(S)-
ethyl-proline (0.50 g, 1.53 mmol), EDC (0.293 g, 1.53 mmol), HOBT
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(0.243 g, 1.53 mmol) and 3-chlorobenzylamine(0.1~7 mL, 1.53 mmol)
were di.ssolved in DMF (5 mL), the pH adjusted to 7 with N-methyl-
morpholine (0.51 mL, 4.6 mmol), and the reaction mixture stirred for
1 ~ hours at ambient temperature. After removing the solvent iM l ac~uo
5 the residue was partitioned between EtOAc and 5% a4ueous NaHCO3.
The organic layer was washed with brine, dried (MgSO4), filtered, and
concentrated to give the title compound after chromatography (SiO2,
EtOAc: hexane, 2:3).
~0 Step I: N-[(t-Butyloxycarbonyl)-pyrrolidin-2(S)-ylmethyl]-3(S)-
ethyl-proline-N-methyl-N-(3-chlorophenylmethyl) amide
N-[(t-Butyloxycarbonyl)-pyrrolidin-2(S)-ylmethyl] -3(S)-
ethyl-proline-N-(3-chlorophenylmethyl) amide (0.175 g, 0.39 mrnol) was
dissolved in dry DMF (4 mL) with stirring at 0~C under Ar, treated with
15 NaH (60% dispersion in mineral oil, 0.023 g, 0.5~ mmol), and after 15
minutes treated with iodomethane (0.029 mL, 0.47 mmol). The reaction
mixture was stirred at 25 ~C. for 2 h, then evaporated to dryness and
partitioned between EtOAc and aqueous saturated NaHCO3 solution.
The organic layer was separated, washed with brine, dried (MgSO4),
20 filtered, and concentrated to dryness to give the title compound.
Step J: (Pyrrolidin-2(S)-ylmethyl)-3(S)-ethyl-proline-N-methyl-N-
(3-chlorophenylmethvl) amide
N-[(t-Butyloxycarbonyl)-pyrrolidin-2(S)-ylmethyl] -3(S)-
25 ethyl-proline-N-methyl-N-(3-chlorophenylmethyl) amide (0.150 g, 0.32
mmol) was dissolved in EtOAc (15 mL), cooled to -20~C. and saturated
with HCI gas. The .solution was stirred at 0~C. for 1 h, then at 25~C. for
1 hour, then concentrated to dryness to give pyrrolidin-2(S)-ylmethyl]-
3(S)-ethyl-proline-N-methyl-N-(3-chlorophenylmethyl) amide which
30 was used without further purification.
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Step K: N-[ 1-(3-[1 H-Irnidazol-4-yl]propionyl)-pyrrolidin-2(S)-
ylmethyl] -3(S)-ethyl-proline-N-(3-chlorophenylmethyl)
amide
(Pyrrolidin-2(S)-ylmethyl)-3(S)-ethyl-proline-N-methyl-N-
5 (3-chlorophenylmethyl) amide (0.070 g, 0.162 mmol), lH-imidazol-4-
yl]propionic acid hydrochloride (0.057 g, 0.324 mmol), EDC (0.062 g,
0.324 mmol), HOBT (0.050 g, 0.324 mmol), and N-methylmorpholine
(0.288 mL, 1.30 mmol) were dissolved in DMF (5 mL) at 25~C. and
stirred for 72 h. The reaction mixture was partitioned between EtOAc
10 and 5% Na2CO3 solution, the organic layer separated, washed with
brine, dried (MgSO4), filtered, and concentrated to dryness to give the
title compound after preparative RP HPLC (Vydac column, 0.1 %
TFA/CH3CN: 0.1% TFA/H20, 95:5 to 5:95 gradient) and Iyophilization.
to give the title compound as the TFA salt.~5 Anal. calcd for C26H36N5O2CI ~ 1.9 CF3C02H- 2.0 H2O:
C, 48.45; H, 5.72; N, 9.48;
found: C, 48.53; H, 5.76; N, 9.08.
FAB MS 486 (M+l)
EXAMPLE 2
Preparation of N-[ 1-(3-[ l H-Imidazol-4-yl]propionyl)-3(S)-
ethylpyrrolidin-2(S)-ylmethyl]-3(S)-ethyl-proline-N-
methyl-N-(3-chlorophenylmethyl) amide
Step A: N-l(tert-Butyloxy)carbonyll-3(S)-ethyl-2(S)-prolinol
3(S)-Ethyl-2(S)-proline hydrochloride (Example 1, Step G)
cont~ining oc-methylbenzylamine (2.0 g, 0.0128 mol) was dissolved in
dioxane ( 10 mL) and H2O (10 mL) with stirring and cooling to 0~C.
30 N,N-diisopropylethylamine (2.2 rnL, 0.0128 mol) and di-tert-butyl-
dicarbonate (2.79 g, 0.0128 mol) were added and stirring was continued
at 23~C for 48 h. The reaction mixture was partitioned between EtOAc
(60 mL) and H2O (30 mL), the organic layer washed with 0.5N NaOH (2
x 40 mL), the aqueous layers combined and washed with EtOAc ( 30 mL)
35 and this layer back-extracted with 0.5 N NaOH (30 mL). The aqueous
layers were combined and carefully acidified at 0~C with lN HCI to pH
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2. This mixture was extracted with EtOAc (3 x 40 mL), the organics
combined, dried (MgSO4), filtered and concentrated to give N-[(te~ t-
Butyloxy)carbonyl--3(S)-ethyl-2(S)-proline as a colorless oil which was
used without purification.
N-[(tert-Butyloxy)carbonyl]-3(S)-ethyl-2(S)-proline (1.6 g, 6.5~ m~mol)
was dissolved in dry THF (10 mL) and treated with borane (lM in THF,
12.5 mL, 12.5 mmol) with stirring at 0 ~C for 2 h, then 23~C for 1 h. The
solution was cooled to 0~C, treated with H2O (20 mL), and extracted
10 with EtOAc (2 x 30 mL). The organics were washed with brine,
saturated NaHCO3, H2O, dried (MgSO4), filtered and concentrated to
give a viscous oil. The oil was dissolved in CH2C12, filtered through dry
SiO2, and the filtrate concentrated to give the title compound as an oil.
lH NMR (CDC13) o 4.97 (d, lH, J= 7 Hz), 3.71 (t, lH, J = 8 Hz), 3.51-
15 3.62 (m, 3H), 3.1~ - 3.26 (m, lH), 1.9 - 2.0 (m, lH), 1.53-1.7 (m, 2H),
1.47 (s, 9H), 1.26 - 1.43 (m, 2H), 0.95 (t, 3H, J = 7 Hz).
Step B: N-~(tert-Butyloxy)carbonyll-3(S)-ethvl-2(S)-prolinal
N-[(te1 t-Butyloxy)carbonyl-3(S)-ethyl-2(S)-prolinol
20 (0.63~ g, 2.78 mmol) and Et3N (1.4 mL, 9.74 mmol) were dissolved
in dry CH2C12 (10 mL) with stirring and cooling to -10~C and treated
dropwise with a solution of S03.pyr (1.33 g, 8.35 mmol) in dry DMSO
(5 mL) keeping the reaction mixture temperature at < 0~C. The mixture
was stirred at 0~C. for 20 minutes then at 5~C for 20 minlltes, and at
25 1 5~C for 1 hour, then poured into ice-cold 0.5 N HCI and the layers
separated. The aqueous layer was extracted with CH2C12 (3 x 20 mL),
organics combined, washed with H2O, aqueous saturated NaHCO3
solution, brine, and dried (Na2SO4). Filtration and concentration to
dryness gave the title compound which was used without purification.
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Step C: N-[(t-Butyloxycarbonyl)-3(S)-ethylpyrrolidin-2~S)-
ylmethyll -3(S)-ethyl-proline
Following the procedure outlined in Example 1, Step G, but
substituting N-[(tert-butyloxy)carbonyl]-3(S)-ethyl-2(S)-prolinal for N-
[(tert-Butyloxy)carbonyl]-2(S)-prolinal the title compound was prepared.
Step D: N-[ l -(3-[1 H-Imidazol-4-yl]propionyl)-3(S)-ethylpyrrolidin-
2(S)-ylmethyll-3(S)-ethyl -proline-N-methyl-N-(3-chloro-
phenylmethyl) amide
Using the procedures de~scribed in Example 1, the title
compound is prepared.
EXAMPLE 3
I ~S Preparation of 1 -~ 1 -(4-Cyanobenzyl)- 1 H-imidazol-5-ylacetyl] pyrrolidin-
2(S)-ylmethyl] -3 (S)-ethyl-proline-N-methyl-N-(3 -chloro-
phenylmethyl) amide
Step A: lH-lmidazole-4- acetic acid methyl ester hydrochloride
A solution of lH-imidazole-4-acetic acid hydrochloride
(4.00g, 24.6 mmol) in methanol (100 ml) was saturated with gaseous
hydrogen chloride. The resulting solution wa~s allowed to stand at room
temperature (RT) for 1 ~ hours. The solvent was evaporated in vacuo to
afford the title compound as a white solid.
lH NMR(CDC13, 400 MHz) ~ 8.85(1H, s),7.45(1H, s), 3.~¢9(2H, s) and
3.75(3H, s) ppm.
Step B: 1 -(Tribenzyl)- 1 H-imidazol-4-ylacetic acid methyl ester
To a solution of 1 H-Imidazole-4- acetic acid methyl ester
hydrochloride (24.85g, 0.141mol) in dimethyl formamide (DMF)
(l l5ml) was added triethylamine (57.2 ml, 0.412mol) and tribenzyl
- bromide(S5.3g, 0.171 mol) and the suspension was stirred for 24 hours.
After this time, the reaction mixture was diluted with ethyl acetate
(EtOAc) (I 1) and water (350 ml). The organic phase was washed with
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saturated. a4ueous. NaHCO3 (350 ml), dried (Na2SO4) and evaporated
in vacuo. The residue was purified by flash chromatography (sio2~ 0-
100% ethyl acetate in hexanes; gradient elution) to provide the title
compound a,s a white solid.
lH ~MR (CDC13, 400 MHz) o 7.35(1H, s), 7.31 (9H, m),7.22(6H, m),
6.76(1H, s), 3.6~(3H, .s) and 3.60(2H, s) ppm.
Step C: 11-(4-Cyanobenzyl)-lH-imidazol-5-yllacetic acid methyl
ester
To a solution of l-(Tribenzyl)-lH-imidazol-4-ylacetic acid
methyl ester (~.00g, 20.9mmol) in acetonitrile (70 ml) was added bromo-
p-toluonitrile (4.10g, 20.92 mmol) and heated at 55~C for 3 hr. After this
time, the reaction was cooled to room temperature and the resulting
imidazolium salt (white precipitate) was collected by filtration. The
filtrate was heated at 55~C for l~s hours. The reaction mixture was
cooled to room temperature and evaporated in vacuo. To the residue was
added EtOAc (70 ml) and the resulting white precipitate collected by
filtration. The precipitated imidazolium salts were combined, suspended
in methanol (100 ml) and heated to reflux for 30 minutes. After this time,
the solvent was removed in vacuo, the resulting residue was suspended
in EtOAc (75ml) and the .solid isolated by filtration and washed (EtOAc).
The solid was treated with saturated aqueous NaHCO3 (300ml) and
CH2C12 (300ml) and stirred at room temperature for 2 hr. The organic
layer was separated, dried (MgSO4) and evaporated in vacuo to afford
the title compound as a white solid:
1HNMR(CDC13, 400 MHz) ~ 7.65(1H, d, J=~Hz), 7.53(1H, s), 7.15(1H,
d, J=f~Hz), 7.04(1H, s), 5.24(2H, s), 3.62(3H, s) and 3.45(2H, s) ppm.
Step D: ~ 1 -(4-Cyanobenzyl)- 1 H-imidazol-5-yllacetic acid
A solution of [ I -(4-cyanobenzyl)- 1 H-imidazol-5-yl]acetic
acid methyl ester (4.44g, 17.4mmol ) in THF (lOOml) and 1 M lithium
hydroxide (17.4 ml, 17.4 mmol) was stirred at RT for 1 ~ hr. I M HCI
(17.4 ml) was added and the THF was removed by evaporation in vacuo.
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The a4ueous ~solution wa~s Iyophilized to afford the title compound
containing lithium chloride as a white ~solid.
1 H NMR(CD30D, 400 MHz) ~ 8.22(1 H, s), 7.74(1 H, d, J-8.4Hz),
7.36(1H, d, J=8.4Hz), 7.15(1H, s), 5.43(2H, s) and 3.49(2H, s) ppm.
s
Step E: I -[1 -(4-Cyanobenzyl)- 1 H-imidazol-5-ylacetyl]
pyrrolidin-2(S )-ylmethyl] -3(S)-ethyl-proline-
N-methyl-N-(3-chlorophenylmethyl) amide
Pyrrolidin-2(S)-ylmethyll -3(S)-ethyl-proline-N-methyl-N-
10 (3-chlorophenylmethyl) amide (0.070 g, 0.1621nmol) (Example 1, Step
J), [1-(4-cyanobenzyl)-lH-imidazol-5-yl]acetic acid ~ LiCI (0.093 g,
0.324 mmol), EDC (0.062 g, 0.324 mmol), HOBT (0.050 g, 0.324 mmol),
and N-methylmorpholine (0.288 mL, 1.30 mmol) were dissolved in DMF
(5 mL) at 25~C. and stirred for 72 h. The reaction mixture was parti-
15 tioned between EtOAc and 5~O Na2C03 solution, the organic layerseparated, washed with brine, dried (MgSO4), filtered, and concentrated
to dryness to give the title compound after preparative RP HPLC (Vydac
column, 0.1 % TFA/CH3CN: 0.1 % TFA/H2O, 95:5 to 5:95 gradient) and
Iyophilization. to give the title compound as the TFA salt. lH NMR
20 (CD30D, 400 MHz) ~ 8.96 and 8.93 (lH, 2 s, 1:2 ratio), 7.7~ - 7.85 (2H,
m), 7.5 - 7.62 (3H, m), 7.2 - 7.4 (4H, m), 5.60 and 5.67 (2H, 2 s, 1 :2
ratio), 4.55 - 4.8 (2H, m), 4.09 - 4.2 (lH, m), 3.82 -4.0 (3H, m), 3.5 - 3.65
(2H, m), 3.35 - 3.49 (2H, m), 3.1~ - 3.3 (lH, m), 3.10 (3H, s), 2.1 - 2.4
(4H, m), 1.92 - 2.09 (3H, m), 1.71 - 1.92 (2H, m), 1.5 - 1.7 (lH, m), 0.96
25 - 1.08 (3H, m) ppm.
FAB MS 587 (M+l )
EXAMPLE 4
30 Preparation of 1 -[1 -(4-Cy~nobenzyl)- 1 H-imidazol-S-ylacetyl] pyrrolidin-
2(S)-ylmethyl] -3(S)-ethyl-proline-N-(3-chlorophenylmethyl)
amide
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Step A: (Pyrrolidin-2(S)-ylmethyl)-3(S)-ethyl-proline-N-(3-
chlorophenylmethyl) amide
N-[(t-Butyloxycarbonyl)-pyrrolidin-2(S)-ylmethyl]-3(S)-
ethyl-proline-N-(3-chlorophenylmethyl) amide (Example 1, Step H)
5 (0.175 g, 0.39 mmol) was dissolved in EtOAc (15 mL), cooled to -20~C.
and ~aturated with HCI gas. The solution was ~tirred at 0~C. for 1 h, then
at 25~C. for 1 h, then concentrated to dryness to give pyrrolidin-2(S)-
ylmethyl]-3(S)-ethyl-proline-N-(3-chlorophenylmethyl) amide which wa,<;
used without further purification.
Step B: 1 -[ I -(4-Cyanobenzyl)- 1 H-imidazol-5-ylacetyl]
pyrrolidin-2(S)-ylmethyl]-3(S)-ethyl-proline-
N-(3-chlorophenylmethvl) amide
Using the procedures described in Example 3, but
substituting pyrrolidin-2(S)-ylmethyl]-3(S)-ethyl-proline-N-(3-
chlorophenylmethyl) amide for pyrrolidin-2(S)-ylmethyl]-3(S)-ethyl-
proline-N-methyl-N-(3-chlorophenylmethyl) amide in Step E, the title
compound was prepared. lH NMR (CD30D, 400 MHz) ~ 8.94 (lH, ~),
7.~s1 (2H, d, J= S Hz), 7.5 - 7.6 (3H, m), 7.12 - 7.3X (4H, m), 5.55 (2H, s),
4.38 - 4.54 (2H, m), 4.02 - 4.1~ (lH, m), 3.75 -4.01 (4H, m), 3.42 - 3.65
(2H, m), 3.42 - 3.65 (2H, m), 3.1 - 3.27 (lH, m), 2.2 - 2.4 (2H, m), 1.6 -
2.2 (7H, m), 1.42 - 1.5~ (lH, m), 1.22 - 1.35 (lH, m), 0.95 - 1.0~ (3H, m)
ppm.
FAB MS 573 (M+l)
EXAMPLE 5
Preparation of 1 -(1 -(4-Nitrobenzyl)- 1 H-imidazol-4-ylacetyl]
pyrrolidin -2(S)-ylmethyl]-3(S)-ethyl-proline-N-methyl-N-
(3-chlorophenylmethyl) amide and 1-[1-(4-Nitrobenzyl)-
lH-imidazol-5-ylacetyl] pyrrolidin -2(S)-ylmethyl]-3(S)-
ethyl-proline-N-methyl-N-(3-chlorophenylmethvl) amide
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Step A: 1-(4-Nitrobenzyl)-lH-imidazol-4-ylacetic acid methyl ester
and 1 -(4-Nitrobenzyl)- I H-imidazol-5-ylacetic acid methyl
ester (3:1 mixture)
To a solution of sodium hydride (60% in mineral oil,
99 mg, 2.5 mmol) in dimethylformamide (2 ml) cooled to 0~C was
added, via cannula, a solution of lH-imidazole-4-acetic acid methyl ester
hydrochloride (200mg, 1.13 mmol) in dimethylformamide (3 ml). This
suspension was allowed to stir at 0~C for 15 min. To this suspension wa,s
added 4-nitrobenzyl bromide (244 mg, 1.13 mmol) and stirred at room
temperature for 2 h. After this time, the mixture was quenched with
saturated. aqueous. sodium bicarbonate (15 ml) and water (20 ml) and
extracted with methylene chloride (2 x 50 ml). The combined organic
extracts were washed with brine (20 ml), dried (MgSO4), filtered and the
solvent was evaporated in vacuo. The residue was purified by flash
chromatography using acetonitrile as eluent to give the title compounds
as a yellow oil.
lH NMR (CDC13, 400 MHz) ~ 8.20 (2H, d, J=8.5 Hz), 7.49 (lH, s), 7.27
(2H, d, J=8.5 Hz), 7.03 (0.25H, s), 6.87 (0.75H, s), 5.28 (O.SH, s), 5.18
(1.5H, s), 3.70 (2.25H, s), 3.65 (1.5H, s), 3.61 (0.75H? s) and 3.44 (0.5H,
20 s) ppm.
Step B: 1-(4-Nitrobenzyl)-lH-imidazol-4-ylacetic acid
hydrochloride and I -(4-Nitrobenzyl)- lH-imidazol-
S-ylacetic acid (3:1mixture)
To a solution of a mixture of I-(4-Nitrobenzyl)-lH-
imidazol-4-ylacetic acid methyl ester and 1-(4-Nitrobenzyl)-lH-
imidazol-5-ylacetic acid methyl ester (3:1mixture, 216 m~, 0.785 rnrnol)
in methanol (3 ml) and tetrahydrofuran (3 ml) under argon was added 1.0
M sodium hydroxide (1.18 ml, 1.18 mmol) and stirred for 1~ h. After this
time, 1.0 N hydrochloric acid (2.36 ml, 2.36 mmol) was added and the
mixture evaporated in vacuo to give the title compounds.
lH NMR (CDC13, 400 MHz) ~ 9.04 (0.75H, s), ~.83 (0.25H, s), 8.28
(2H, d, J=8.8 Hz), 7.61 (2H, d, J=8.~ Hz), 7.54 (0.75H, s),7.43 (0.25H?
s), 5.61 (0.5H, s), 5.58 (1.5H, s), 3.84 (0.5H, s) and 3.8~ (1.5H? s) ppm.
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Step C~ (4-Nitrobenzyl)- 1 H-imidazol-4-ylacetyl]
pyrrolidin -2(S)-ylmethyl]-3(S)-ethyl-proline-N-methyl-N-
(3-chlorophenylmethyl) amide and 1-[1-(4-Nitrobenzyl)-
lH-imidazol-5-ylacetyl] pyrrolidin -2(S)-ylmethyl]-3(S)-
ethyl-proline-N-(3-chlorophenylmethyl) amide
Using the procedure.~ outlined in Examples 3 and 4, the title
compound~ are prepared.
The following compounds are prepared in a ~imilar manner:
I -(1 -(4-Nitrobenzyl)- 1 H-imidazol-4-ylacetyl] pyrrolidin -2(S)-
ylmethyl]-3(S)-ethyl-proline-N-(3-chlorophenylmethyl) amide
1-[1 -(4-Nitrobenzyl)- 1 H-imidazol-5-ylacetyll pyrrolidin -2(S)-
ylmethyll-3(S)-ethyl-proline-N-(3-chlorophenylmethyl) amide
-
1 -(1 -(4-Methoxybenzyl)- 1 H-imidazol-5-ylacetyl)pyrrolidin-2(S)-
ylmethyll-3(S)-ethyl-proline-N-methyl-N-(3-chlorophenylmethyl) amide
20 1-(1 -(4-Methoxybenzyl)- 1 H-imidazol-5-ylacetyl)pyrrolidin-2(S)-
vlmethyll-3(S)-ethyl-proline-N-(3-chlorophenylmethyl) amide
1-(1 -(2-Naphthylmethyl)- 1 H-imidazol -5-ylacetyl]pyrrolidin-2(S)-
ylmethyll-3(S)-ethyl-proline-N-methyl-N-(3-chlorophenylmethyl) amide
1-(1 -(2-Naphthylmethyl)- 1 H-imidazol-5-ylacetyl]pyrrolidin-2(S)-
ylmethyll-3(S)-ethyl-proline-N-(3-chlorophenylmethyl) amide
EXAMPLE 6
Preparation of 1-(1-(1 -Farnesyl)- 1 H-imidazol-S-ylacetyl)-pyrrolidin-2(S)-ylmethyll -3(S)-ethyl -proline-N-methyl-N-(3-
chlorophenylmethyl) amide
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Step A: l-(1-Farnesyl)-lH-imidazol-5-ylacetic acid methyl e,ster
To a solution of l-(tribenzyl)-lH-imidazol-4-ylacetic acid
methyl e.ster (200 mg, 0.523 mmol) in acetonitrile (5 ml) was added trans,
trans-farnesyl bromide (156 ,ul, 0.575 mmol) and heated at 55~C for 16 h.
5 After this time, the reaction was heated at 80~C for 3 h and then the
reaction mixture was evaporated in vacuo. The residue was dissolved in
methanol (5 ml ) and heated to reflux for 30 minutes and then evaporated
in vacuo. The residue was purified by flash chromatography (2-4%
methanol/methylene chloride gradient elution) to provide the title
10 compound.
lH NMR (CDC13, 400 MHz) ~ 7.50 (lH, s), 6.92 (lH, s), 5.24 (lH, t,
J-5.9 Hz), 5.09 (2H, m), 4.49 (2H, d, J=6.9 Hz), 3.69 (3H, s), 3.60 (2H,
s), 1.91-2.15 (8H, m), 1.72 (3H, s), 1.65 (3H, s), 1.59 (3H, s) and 1.57
(3H, s) ppm.
Step B: 1 -(1 -(1 -Farnesyl)- 1 H-imidazol-S-ylacetyl)-pyrrolidin-
2(S)-ylmethyl]-3(S)-ethyl-proline-N-methyl-N-(3-
chlorophenylmethyl) amide
Following the procedure described in Example 5, but using
20 1 -farnesyl- 1 H-imidazol-5-ylacetic acid methyl ester described in Step A
in place of 1-(4-nitrobenzyl)-lH-imidazol-S-ylacetic acid methyl ester
provides the title compound.
1 -(1 -(1 -Geranyl)- 1 H-imidazol-5-ylacetyl)-pyrrolidin-2(S)-ylmethyl] -
25 3(S)-ethyl-proline-N-methyl-N-(3-chlorophenylmethyl) amide is
prepared in a similar manner.
EXAMPLE 7
30 In vitro inhibition o~ ras farnesyl transfera.se
Assays offarnesyl-protein tran~èrase. 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.
2~5: 14701 - 14704 (1990), Pompliano, et al., Biochemist~y 31 :3800
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( l 992) and Gibbs et al., PNAS U.S.A. 8~:6630-6634 ( l 9g9), respectively.
Bovine FPTase wa.s assayed in a volume of l00 !ll containing l00 mM
N-(2-hydroxy ethyl) piperazine-N'-(2-ethane .~iulfonic acid) (HEPES), pH
7.4, 5 mM MgCl2, 5 mM dithiothreitol (DTT), l00 mM [3H]-farnesyl
5 diphosphate ([3H]-FPP; 740 CBq/mmol, New ~ngland Nuclear), 650 nM
Ras-CVLS and 10 ~g/ml FPTase at 31 ~C for 60 min. Reaction,s were
initiated with FPTase and stopped with 1 ml of l.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
l0 ~-plate counter. The assay 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
l 5 radioactivity in the presence of the test compound when compared to
the amount of incorporation in the absence of the test compound.
Human FPTase was prepared as described by Omer et al.,
Biochemistry 32:5167-5l76 (1993). Human FPTase activity was assayed
as described above with the exception that 0.1% (w/v) polyethylene
20 glycol 20,000, 10 ~lM ZnCl2 and l00 nM Ras-CVIM were added to the
reaction mixture. Reactions were performed for 30 min., stopped with
100 !11 of 30% (v/v) trichloroacetic acid (TCA) in ethanol and processed
as described above for the bovine enzyme.
The compounds of the instant invention were tested for
25 inhibitory activity against human FPTase by the assay described above
and were found to have IC50 of < 10 ~M.
EXAMPLE
30 In viv(~ ras farnesylation assay
The cell line used in this assay is a v-ras line derived from
either Ratl or NIH3T3 cell~;, which expressed viral Ha-ras p21. The
assay is performed essentially as described in DeClue, J.E. et al..
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CancerResea~ch 51:712-717, (1991). Cell.s in 10 cm dishes at 50-75%
confluency are treated with the test compound (final concentration of
solvent, methanol or dimethyl sulfoxide, is 0.1 %). After 4 hours at 37~C,
the cells are labeled in 3 ml methionine-free DMEM supple-meted with
10% regular DMEM, 2% fetal bovine serum and 400 mCiL35S]
methionine (1000 Ci/mmol). After an additional 20 hours, the cells are
Iysed 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 counts are bought to I ml with IP buffer (Iysis buffer
lacking DTT) and immunoprecipitated with the ras-specific monoclonal
antibody Y13-259 (Furth, M.E. etal., J. Vi~ol. 43:294-304, (19~2)).
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/1 % Triton X- 100Ø5%
deoxycholate/0.1 %/SDS/0.1 M NaCI) boiled in SDS-PAGE sample
buffer and loaded on 13% acrylamide gels When the dye front reached
the bottom, the gel is fixed, soaked in Enlightening, dried and auto-
radiographed. The intensities of the bands corresponding to farnesylated
and nonfarnesylated ras protein~s are compared to determine the percent
inhibition of farnesyl transfer to protein.
EXAMPLE 9
ln vil~o ~rowth inhibition assay
To determine the biological conse4uence~s of FPTase
inhibition, the effect of the compounds of the instant invention on the
30 anchorage-independent growth of Ratl cells transformed with either a
v-ras, v-~ af, or v-mos oncogene is tested. Cells transforrned 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
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are seeded at a den.sity 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
5 appropriate concentration of the instant compound (dis~solved 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.
