CA2201347A1 - Inhibitors of farnesyl-protein transferase - Google Patents
Inhibitors of farnesyl-protein transferaseInfo
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- CA2201347A1 CA2201347A1 CA 2201347 CA2201347A CA2201347A1 CA 2201347 A1 CA2201347 A1 CA 2201347A1 CA 2201347 CA2201347 CA 2201347 CA 2201347 A CA2201347 A CA 2201347A CA 2201347 A1 CA2201347 A1 CA 2201347A1
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- substituted
- unsubstituted
- aryl
- heterocycle
- cycloalkyl
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Abstract
The present invention comprises dipeptide analogs that inhibit the farnesylation of Ras. These farnesyl-protein transferase inhibitors are characterized by the inclusion of a cyclic amine in the backbone of the dipeptide. Further contained in this invention are chemotherapeutic compositions containing these farnesyl transferase inhibitors and methods for their production.
Description
W03'~v3~20 22Q1 3~ ? PCT/US95/12284 , . . .
TITLE OF THE INVENTION
INHIBITORS OF FARNESYL-PROTEIN TRANSFERASE
BACKGROUND OF THE INVENTION
The Ras gene is found activated in many human cancers, including colorectal carcinoma, exocrine pancreatic carcinoma, and myeloid leukemias. Biological and biochemical studies of Ras action indicate that Ras functions like a G-regulatory protein, since Ras must be localized in the plasma membrane and must bind with GTP in order to transform cells (Gibbs, J. et al., Microbiol. Rev. 53:171-286 (1989). Forms of Ras in cancer cells have mutations that distinguish the protein from Ras in normal cells.
At least 3 post-tr~n~l~tional modifications are involved with Ras membrane loc~li7~tion, and all 3 modifications occur at the C-terminus of Ras. The Ras C-terminlls contains a sequence motif termed a "CAAX" or "Cys-Aaa1-Aaa2-Xaa" box (Aaa is an aliphatic amino acid, the Xaa is any amino acid) (Willumsen et al., Nature 310:583-586 (1984)). Other proteins having this motif include the Ras-related GTP-binding proteins such as Rho, fungal m~tinp factors, the nuclear l~min~, and the gamma subunit of transducin.
Farnesylation of Ras by the isoprenoid farnesyl pyrophosphate (FPP) occurs in vivo on Cys to form a thioether linkage (Hancock et al., Cell 57: 1167 (1989); Casey et al., Proc. Natl. Acad.
Sci. USA 86:8323 (1989)). In addition, Ha-Ras and N-Ras are palmitoylated via formation of a thioester on a Cys residue near a C-terminal Cys farnesyl acceptor (Gutierrez et al., EMBO J. 8:1093-1098 (1989); Hancock et al., Cell 57:1167-1177 (1989)). Ki-Ras lacks the p~lmit~te acceptor Cys. The last 3 amino acids at the Ras C-termin~l end are removed proteolytically, and methyl esterification occurs at the new C-te"lli~ ,c (Hancock et al., ibi~). Fungal m~ting factor and m~mm~ n nuclear l~min~ undergo identical modification steps (Anderegg et al., J. Biol. Chem. 263:18236 (1988); Farnsworth et al., J. Biol. Chem. 264:20422 (1989)).
WO ~GI'~20 . PCT/US~S112284 ~Q~3~7 2-~ hibition of Ras farnesylation in vivo has been demonstrated with lovastatin (Merck & Co., Rahway, NJ) and compactin (Hancock et al., ibid; Casey et al., ibid; Schafer et al., Science 245:379 (1989)). ~hese drugs inhibit HMG-CoA reductase, 5 the rate limiting enzyme for the production of polyisoprenoids and the farnesyl pyrophosphate precursor. It has been shown that a farnesyl-protein transferase using farnesyl pyrophosphate as a precursor is responsible for Ras farnesylation. (Reiss et al., Cell, 62:81-88 (1990), Schaber et al., J. Biol. Chem., 265:14701-14704 (1990); Schafer et al., 10 Science, 249: 1133-1139 (1990); Manne et al., Proc. Natl. Acad. Sci USA, 87:7541-7545 (1990)).
Inhibition of farnesyl-protein transferase and, thereby, of farnesylation of the Ras protein, blocks the ability of Ras to transform normal cells to cancer cells. The compounds of the invention irlhibit Ras farnesylation and, thereby, generate soluble Ras which, as indicated infra, can act as a domin~nt negative inhibitor of Ras function. While soluble Ras in cancer cells can become a domin~nt negative inhibitor, soluble Ras in normal cells would not be an inhibitor.
A cytosol-localized (no Cys-Aaa1-Aaa2-Xaa box membrane domain present) and activated (impaired GTPase activity, staying bound to GTP) form of Ras acts as a domin~nt negative Ras inhibitor of membrane-bound Ras function (Gibbs et al., Proc. Natl.
Acad. Sci. USA 86:6630-6634(1989)). Cytosollocalized forms of Ras with normal GTPase activity do not act as inhibitors. Gibbs et al., ibid, showed this effect in Xenopus oocytes and in m~mm~ n cells.
A-lmini.~tration of compounds of the invention to block Ras farnesylation not only decreases the amount of Ras in the membrane but also generates a cytosolic pool of Ras. In tumor cells having activated Ras, the cytosolic pool acts as another antagonist of membrane-bound Ras function. In normal cells having normal Ras, the cytosolic pool of Ras does not act as an antagonist. In the absence of complete inhibition of farnesylation, other farnesylated proteins are able to continue with their functions.
WO ~ 20 PCTIUS95/12284 ~OI3q~
.; , , Farnesyl-protein transferase activity may be reduced or completely inhibited by adjusting the compound dose. Reduction of farnesyl-protein transferase enzyme activity by adjusting the compound dose would be useful for avoiding possible undesirable 5 side effects resulting from interference with other metabolic processes which utilize the enzyme.
These compounds and their analogs are inhibitors of farnesyl-protein transferase. Farnesyl-protein transferase utilizes farnesyl pyrophosphate to covalently modify the Cys thiol group of the Ras CAAX box with a farnesyl group. Inhibition of farnesyl pyrophosphate biosynthesis by inhibiting HMG-CoA reductase blocks Ras membrane loc~li7~tion in vivo and inhibits Ras function.
Inhibition of farnesyl-protein transferase is more specific and is attended by fewer side effects than is the case for a general inhibitor 15 of isoprene biosynthesis.
Previously, it has been demonstrated that tetrapeptides cont~inin~ cysteine as an amino termin~l residue with the CAAX
sequence inhibit Ras farnesylation (Schaber et al., ibid; Reiss et. al., ibid; Reiss et al., PNAS, 88:732-736 (1991)). Previously described 20 CAlA2X-type FPTase inhibitors cQntain acyclic amino acids in the second position. Incorporation of proline in the Al position in such inhibitors has been shown to be the least well tolerated amino acid substitution in that position (Reiss et al., PNAS (1991)). Such inhibitors may inhibit while serving as alternate substrates for the Ras
TITLE OF THE INVENTION
INHIBITORS OF FARNESYL-PROTEIN TRANSFERASE
BACKGROUND OF THE INVENTION
The Ras gene is found activated in many human cancers, including colorectal carcinoma, exocrine pancreatic carcinoma, and myeloid leukemias. Biological and biochemical studies of Ras action indicate that Ras functions like a G-regulatory protein, since Ras must be localized in the plasma membrane and must bind with GTP in order to transform cells (Gibbs, J. et al., Microbiol. Rev. 53:171-286 (1989). Forms of Ras in cancer cells have mutations that distinguish the protein from Ras in normal cells.
At least 3 post-tr~n~l~tional modifications are involved with Ras membrane loc~li7~tion, and all 3 modifications occur at the C-terminus of Ras. The Ras C-terminlls contains a sequence motif termed a "CAAX" or "Cys-Aaa1-Aaa2-Xaa" box (Aaa is an aliphatic amino acid, the Xaa is any amino acid) (Willumsen et al., Nature 310:583-586 (1984)). Other proteins having this motif include the Ras-related GTP-binding proteins such as Rho, fungal m~tinp factors, the nuclear l~min~, and the gamma subunit of transducin.
Farnesylation of Ras by the isoprenoid farnesyl pyrophosphate (FPP) occurs in vivo on Cys to form a thioether linkage (Hancock et al., Cell 57: 1167 (1989); Casey et al., Proc. Natl. Acad.
Sci. USA 86:8323 (1989)). In addition, Ha-Ras and N-Ras are palmitoylated via formation of a thioester on a Cys residue near a C-terminal Cys farnesyl acceptor (Gutierrez et al., EMBO J. 8:1093-1098 (1989); Hancock et al., Cell 57:1167-1177 (1989)). Ki-Ras lacks the p~lmit~te acceptor Cys. The last 3 amino acids at the Ras C-termin~l end are removed proteolytically, and methyl esterification occurs at the new C-te"lli~ ,c (Hancock et al., ibi~). Fungal m~ting factor and m~mm~ n nuclear l~min~ undergo identical modification steps (Anderegg et al., J. Biol. Chem. 263:18236 (1988); Farnsworth et al., J. Biol. Chem. 264:20422 (1989)).
WO ~GI'~20 . PCT/US~S112284 ~Q~3~7 2-~ hibition of Ras farnesylation in vivo has been demonstrated with lovastatin (Merck & Co., Rahway, NJ) and compactin (Hancock et al., ibid; Casey et al., ibid; Schafer et al., Science 245:379 (1989)). ~hese drugs inhibit HMG-CoA reductase, 5 the rate limiting enzyme for the production of polyisoprenoids and the farnesyl pyrophosphate precursor. It has been shown that a farnesyl-protein transferase using farnesyl pyrophosphate as a precursor is responsible for Ras farnesylation. (Reiss et al., Cell, 62:81-88 (1990), Schaber et al., J. Biol. Chem., 265:14701-14704 (1990); Schafer et al., 10 Science, 249: 1133-1139 (1990); Manne et al., Proc. Natl. Acad. Sci USA, 87:7541-7545 (1990)).
Inhibition of farnesyl-protein transferase and, thereby, of farnesylation of the Ras protein, blocks the ability of Ras to transform normal cells to cancer cells. The compounds of the invention irlhibit Ras farnesylation and, thereby, generate soluble Ras which, as indicated infra, can act as a domin~nt negative inhibitor of Ras function. While soluble Ras in cancer cells can become a domin~nt negative inhibitor, soluble Ras in normal cells would not be an inhibitor.
A cytosol-localized (no Cys-Aaa1-Aaa2-Xaa box membrane domain present) and activated (impaired GTPase activity, staying bound to GTP) form of Ras acts as a domin~nt negative Ras inhibitor of membrane-bound Ras function (Gibbs et al., Proc. Natl.
Acad. Sci. USA 86:6630-6634(1989)). Cytosollocalized forms of Ras with normal GTPase activity do not act as inhibitors. Gibbs et al., ibid, showed this effect in Xenopus oocytes and in m~mm~ n cells.
A-lmini.~tration of compounds of the invention to block Ras farnesylation not only decreases the amount of Ras in the membrane but also generates a cytosolic pool of Ras. In tumor cells having activated Ras, the cytosolic pool acts as another antagonist of membrane-bound Ras function. In normal cells having normal Ras, the cytosolic pool of Ras does not act as an antagonist. In the absence of complete inhibition of farnesylation, other farnesylated proteins are able to continue with their functions.
WO ~ 20 PCTIUS95/12284 ~OI3q~
.; , , Farnesyl-protein transferase activity may be reduced or completely inhibited by adjusting the compound dose. Reduction of farnesyl-protein transferase enzyme activity by adjusting the compound dose would be useful for avoiding possible undesirable 5 side effects resulting from interference with other metabolic processes which utilize the enzyme.
These compounds and their analogs are inhibitors of farnesyl-protein transferase. Farnesyl-protein transferase utilizes farnesyl pyrophosphate to covalently modify the Cys thiol group of the Ras CAAX box with a farnesyl group. Inhibition of farnesyl pyrophosphate biosynthesis by inhibiting HMG-CoA reductase blocks Ras membrane loc~li7~tion in vivo and inhibits Ras function.
Inhibition of farnesyl-protein transferase is more specific and is attended by fewer side effects than is the case for a general inhibitor 15 of isoprene biosynthesis.
Previously, it has been demonstrated that tetrapeptides cont~inin~ cysteine as an amino termin~l residue with the CAAX
sequence inhibit Ras farnesylation (Schaber et al., ibid; Reiss et. al., ibid; Reiss et al., PNAS, 88:732-736 (1991)). Previously described 20 CAlA2X-type FPTase inhibitors cQntain acyclic amino acids in the second position. Incorporation of proline in the Al position in such inhibitors has been shown to be the least well tolerated amino acid substitution in that position (Reiss et al., PNAS (1991)). Such inhibitors may inhibit while serving as alternate substrates for the Ras
2 farnesyl-transferase enzyme, or may be purely competitive inhibitors (U.S. Patent 5,141,851, University of Texas).
It has also been demonstrated that certain inhibitors of farnesyl-protein transferase selectively block the processing of Ras oncoprotein intracellularly (N.E. Kohl et al., Science, 260:1934-1937 30 (1993) and G.L. James et al., Science, 260:1937-1942 (1993)).
Recently, it has been shown that an inhibitor of farnesyl-protein transferase blocks the growth of ras-dependent tumors in nude mice (N.E. Kohl et al., Proc. Natl. Acad. Sci U.S~., 91 :9141 -9145 (1994) ).
WO ~G,r~20 PCT/USS~5112284 22~134~
Inhibitors of Ras farnesyl-protein transferase (FPTase) have been described in two general classes. The first are analogs of farnesyl diphosphate (FPP), while the second class of inhibitors is related to the protein substrate for the enzyme, Ras. Almost all of the peptide derived inhibitors that have been described are cysteine cont~ining molecules that are related to the CAAX motif that is the signal for protein prenylation. The exception to this generalization is a class of natural products known as the pepticinn~min~ (Omura, et al., J. Antibiotics 46:222 (1993)).
It is, therefore, an object of this invention to develop tetrapeptide-based compounds which incorporate a cyclic amino acid in the second position, and which will inhibit farnesyl transferase and the post-translational function~li7~tion of the oncogene Ras protein. It is a further object of this invention to develop chemotherapeutic compositions cont~ining the compounds of this invention and methods for producing the compounds of this invention.
SUM~ARY OF THE INVENTION
The present invention comprises dipeptide analogs that inhibit the farnesylation of Ras. These compounds differ from those previously described as preferred inhibitors of Ras farnesyl transferase in that, in addition to being dipeptide-like, they incorporate a cyclic amine moiety in the position coresponding to the second amino acid of the dipeptide-like structure. Further contained in this invention are chemotherapeutic compositions cont~ining these farnesyl transferase inhibitors and methods for their production.
f 2201~47 - =:
The compounds of this invention are illustrated by the form~
Z
HS ~ 4 X~y~ R3 R1HN /~ </
(C~H2)t R2a~ R2b I
DETAILED DESCRIPTION OF THE INVENTION
The compounds of this invention inhibit the farnesylation of Ras. In a first embodiment of this invention, the Ras farnesyl transferase inhibitors are illustrated by the formula I:
HS ~ y, R1 HN ~
(C~H2)t 2 o R2a~ ~ R2b I
wherein:
R 1 is selected from:
a) hydrogen, b) R5S(0)2-, RSC(O)-, (R5~2NC(o)- or R60C(O)-, and c) C 1 -C6 alkyl unsubstituted or substituted by aryl, heterocyclic, cycloalkyl, alkenyl, alkynyl, R50-, R5S(o)m-~ R5C(o)NR5-, CN, (R5)2N-C(NR5)-, RSC(O)-, R50C(o)-, N3, -N(R5)2, or R60C(O)NR5-;
R2a and R2b are independently selected from:
a) hydrogen, WO ~b~/0!~3~0 PCT/US~5/12284 220~34~ ~
b) Cl-C6 alkyl unsubstituted or substituted by aryL
heterocycle, cycloaLkyl, alkenyl, R50-, R5S(o)m R5C(o)NR5-, CN, (R5)2N-C(NR5)-, R5C(o)-, R50C(o)-, N3, -N(R5)2~ or R6oC(o)NR5-, and c) aryl, heterocycle, cycloalkyl, alkenyl, R50-, R5S(o)m-~ R5C(o)NRS-, CN, N02, (R5)2N-C(NRS)-, R5C(o)-, R50C(o)-, N3, -N(R5)2, or R6oC(o)NR5-, R3 is selected from:
o a) unsubstituted or substituted aryl, b) unsubstituted or substituted heterocycle, c) unsubstituted or substituted cycloalkyl, and d) Cl-C6 alkyl substituted with hydrogen or an unsubstituted or substituted group selected from aryl, heterocycle and cycloalkyl;
X-Y is ~4a a) ~sss~N~ss R4b b) \sss~N~ss c)~ ~0~5s WO ~G/'~20 PCTIUS95/12284 22~f34~ `
()m d) ~sss~S~ss e) ~555~ , or f) -CH2-CH2-;
R4a is selected from a) hydrogen, b) unsubstituted or substituted aryl, c) unsubstituted or substituted heterocycle, d) unsubstituted or substituted cycloalkyl, and e) C 1 -C6 alkyl substituted with hydrogen or an unsubstituted or substituted group selected from aryl, heterocycle and cycloalkyl;
R4b iS selected from a) hydrogen, b) unsubstituted or substituted aryl, c) unsubstituted or substituted heterocycle, d) unsubstituted or substituted cycloalkyl, e) Cl-C6 alkyl substituted with hydrogen or~an unsubstituted or substituted group selected from aryl, heterocycle and cycloalkyl, f) a carbonyl group which is bonded to an unsubstituted or substituted group selected from aryl, heterocycle, cycloalkyl and Cl-C6 alkyl substituted with hydrogen or an unsubstituted or substituted group selected from aryl, heterocycle and cycloalkyl, and g) a sulfonyl group which is bonded to an unsubstituted or substituted group selected from aryl, heterocycle, cycloalkyl and Cl-C6 alkyl substituted with hydrogen or an unsubstituted or substituted group selected from aryl, heterocycle and cycloaL~yl;
5 R5 is independently selected from hydrogen, C1-C6 alkyl and aryl;
R6 is independently selected from C1-C6 alkyl and aryl;
Z is independently H2 or 0;
m is 0, 1 or 2, provided that m is O when R5 = hydrogen;
nisO,1,2,30r4; and t is 3, 4 or 5;
or the pharmaceutically acceptable salts thereof.
In a more preferred embodiment of this invention, the Ras farnesyl transferase inhibitors are illustrated by the formula I:
HS--y~ y, R2a~ R2b wherein:
Rl is selected from:
a) hydrogen, b) R5S(0)2-, RSC(O)-, (R5)2NC(o)- or R60C(O)-, and
It has also been demonstrated that certain inhibitors of farnesyl-protein transferase selectively block the processing of Ras oncoprotein intracellularly (N.E. Kohl et al., Science, 260:1934-1937 30 (1993) and G.L. James et al., Science, 260:1937-1942 (1993)).
Recently, it has been shown that an inhibitor of farnesyl-protein transferase blocks the growth of ras-dependent tumors in nude mice (N.E. Kohl et al., Proc. Natl. Acad. Sci U.S~., 91 :9141 -9145 (1994) ).
WO ~G,r~20 PCT/USS~5112284 22~134~
Inhibitors of Ras farnesyl-protein transferase (FPTase) have been described in two general classes. The first are analogs of farnesyl diphosphate (FPP), while the second class of inhibitors is related to the protein substrate for the enzyme, Ras. Almost all of the peptide derived inhibitors that have been described are cysteine cont~ining molecules that are related to the CAAX motif that is the signal for protein prenylation. The exception to this generalization is a class of natural products known as the pepticinn~min~ (Omura, et al., J. Antibiotics 46:222 (1993)).
It is, therefore, an object of this invention to develop tetrapeptide-based compounds which incorporate a cyclic amino acid in the second position, and which will inhibit farnesyl transferase and the post-translational function~li7~tion of the oncogene Ras protein. It is a further object of this invention to develop chemotherapeutic compositions cont~ining the compounds of this invention and methods for producing the compounds of this invention.
SUM~ARY OF THE INVENTION
The present invention comprises dipeptide analogs that inhibit the farnesylation of Ras. These compounds differ from those previously described as preferred inhibitors of Ras farnesyl transferase in that, in addition to being dipeptide-like, they incorporate a cyclic amine moiety in the position coresponding to the second amino acid of the dipeptide-like structure. Further contained in this invention are chemotherapeutic compositions cont~ining these farnesyl transferase inhibitors and methods for their production.
f 2201~47 - =:
The compounds of this invention are illustrated by the form~
Z
HS ~ 4 X~y~ R3 R1HN /~ </
(C~H2)t R2a~ R2b I
DETAILED DESCRIPTION OF THE INVENTION
The compounds of this invention inhibit the farnesylation of Ras. In a first embodiment of this invention, the Ras farnesyl transferase inhibitors are illustrated by the formula I:
HS ~ y, R1 HN ~
(C~H2)t 2 o R2a~ ~ R2b I
wherein:
R 1 is selected from:
a) hydrogen, b) R5S(0)2-, RSC(O)-, (R5~2NC(o)- or R60C(O)-, and c) C 1 -C6 alkyl unsubstituted or substituted by aryl, heterocyclic, cycloalkyl, alkenyl, alkynyl, R50-, R5S(o)m-~ R5C(o)NR5-, CN, (R5)2N-C(NR5)-, RSC(O)-, R50C(o)-, N3, -N(R5)2, or R60C(O)NR5-;
R2a and R2b are independently selected from:
a) hydrogen, WO ~b~/0!~3~0 PCT/US~5/12284 220~34~ ~
b) Cl-C6 alkyl unsubstituted or substituted by aryL
heterocycle, cycloaLkyl, alkenyl, R50-, R5S(o)m R5C(o)NR5-, CN, (R5)2N-C(NR5)-, R5C(o)-, R50C(o)-, N3, -N(R5)2~ or R6oC(o)NR5-, and c) aryl, heterocycle, cycloalkyl, alkenyl, R50-, R5S(o)m-~ R5C(o)NRS-, CN, N02, (R5)2N-C(NRS)-, R5C(o)-, R50C(o)-, N3, -N(R5)2, or R6oC(o)NR5-, R3 is selected from:
o a) unsubstituted or substituted aryl, b) unsubstituted or substituted heterocycle, c) unsubstituted or substituted cycloalkyl, and d) Cl-C6 alkyl substituted with hydrogen or an unsubstituted or substituted group selected from aryl, heterocycle and cycloalkyl;
X-Y is ~4a a) ~sss~N~ss R4b b) \sss~N~ss c)~ ~0~5s WO ~G/'~20 PCTIUS95/12284 22~f34~ `
()m d) ~sss~S~ss e) ~555~ , or f) -CH2-CH2-;
R4a is selected from a) hydrogen, b) unsubstituted or substituted aryl, c) unsubstituted or substituted heterocycle, d) unsubstituted or substituted cycloalkyl, and e) C 1 -C6 alkyl substituted with hydrogen or an unsubstituted or substituted group selected from aryl, heterocycle and cycloalkyl;
R4b iS selected from a) hydrogen, b) unsubstituted or substituted aryl, c) unsubstituted or substituted heterocycle, d) unsubstituted or substituted cycloalkyl, e) Cl-C6 alkyl substituted with hydrogen or~an unsubstituted or substituted group selected from aryl, heterocycle and cycloalkyl, f) a carbonyl group which is bonded to an unsubstituted or substituted group selected from aryl, heterocycle, cycloalkyl and Cl-C6 alkyl substituted with hydrogen or an unsubstituted or substituted group selected from aryl, heterocycle and cycloalkyl, and g) a sulfonyl group which is bonded to an unsubstituted or substituted group selected from aryl, heterocycle, cycloalkyl and Cl-C6 alkyl substituted with hydrogen or an unsubstituted or substituted group selected from aryl, heterocycle and cycloaL~yl;
5 R5 is independently selected from hydrogen, C1-C6 alkyl and aryl;
R6 is independently selected from C1-C6 alkyl and aryl;
Z is independently H2 or 0;
m is 0, 1 or 2, provided that m is O when R5 = hydrogen;
nisO,1,2,30r4; and t is 3, 4 or 5;
or the pharmaceutically acceptable salts thereof.
In a more preferred embodiment of this invention, the Ras farnesyl transferase inhibitors are illustrated by the formula I:
HS--y~ y, R2a~ R2b wherein:
Rl is selected from:
a) hydrogen, b) R5S(0)2-, RSC(O)-, (R5)2NC(o)- or R60C(O)-, and
3 c) Cl-C6 aLkyl unsubstituted or substituted by aryl, heterocyclic, cycloalkyl, alkenyl, alkynyl, R50-, R5S(o)m-~ RSC(O)NR5-, CN, (R5)2N-C(NR5)-, R5C(o)-, RSOC(O)-, N3, -N(R5)2, or R60C(O)NR5-;
~ 2201347 C ~
t ' ., ;-, -R2a and R2b are independently selected from:
a) hydrogen, b) Cl-C6 alkyl unsubstituted or substituted by aryl, heterocycle, cycloalkyl, alkenyl, R50-, R5S(o)m-, R5C(o)NR5-, CN, (R5)2N-C(NR5)-, R5C(o)-, R50C(o)-, N3, -N(R5)2, or R6oC(o)NR5-, and c) aryl, heterocycle, cycloalkyl, alkenyl, RS0-, R5S(o)m-~ R5C(o)NR5-, CN, N02, (R5)2N-C(NR5)-, R5C(o)-, R50C(o)-, N3, -N(R5)2, or R60C(O)NRS-, R3 is selected from:
a) unsubstituted or substituted aryl, b) unsubstituted or substituted heterocycle, c) unsubstituted or substituted cycloalkyl, and d) C1-C6 alkyl substituted with an unsubstituted or substituted group selected from aryl, heterocycle and cycloalkyl;
X-Y is R4a a) ~sss~Nsss . or o R4b b)\~s~N~ss R4a is selected from a) hydrogen, b) unsubstituted or substituted aryl, c) unsubstituted or substituted heterocycle, d) unsubstituted or substituted cycloaL~yl, and WO 9CI'u5~10 PCT/US95/12284 22013~7 e) C 1 -C6 aL~yl substituted with hydrogen or an unsubstituted or substituted group selected from aryl, heterocycle and cycloalkyl;
wherein heterocycle is selected from pyrrolidinyl, imidazolyl, pyridinyl, thiazolyl, pyridonyl, 2-oxopiperidinyl, indolyl, quinolinyl, isoquinolinyl, and thienyl;
R4b is selected from o a) hydrogen, b) unsubstituted or substituted aryl, c) unsubstituted or substituted heterocycle, d) unsubstituted or substituted cycloaL~yl, e) C 1 -C6 aL~yl substituted with hydrogen or an unsubstituted or substituted group selected from aryl, heterocycle and cycloalkyl, f) a carbonyl group which is bonded to an unsubstituted or substituted group selected from aryl, heterocycle, cycloalkyl and Cl -C6 alkyl substituted with hydrogen or an unsubstituted or substituted group selected from aryl, heterocycle and cycloalkyl, and g) a sulfonyl group which is bonded to an unsubstituted or substituted group selected from aryl, heterocycle, cycloaL~yl and Cl -C6 alkyl substituted with hydrogen or an unsubstituted or substituted group selected from aryl, heterocycle and cycloalkyl;
wherein heterocycle is selected from pyrrolidinyl, imidazolyl, pyridinyl, thiazolyl, pyridonyl, 2 oxopiperidinyl, indolyl, quinolinyl, isoquinolinyl, and thienyl;
R5 is independently selected from hydrogen, Cl-C6 alkyl and aryl;
R6 is independently selected from Cl-C6 alkyl and aryl;
WO~GI'~ O PCT/US95112284 22013~7 Z is independently H2 or 0;
m is 0, 1 or 2, provided that m is 0 when R5 = hydrogen;
nis 0,1,2,30r4;and t is 3, 4 or 5;
or the pharmaceutically acceptable salts thereof.
The preferred compounds of this invention are as follows:
N-[2(R)-Amino-3 -mercaptopropyl] -L-proline-2,3 -dichlorobenzylamide N-[2(R)-Amino-3-mercaptopropyl]-L-proline-1-naphthylmethyl amide N-[2(R)-Amino-3-mercaptopropyl] -L-pipecolyl-2,3-dichlorobenzamide N-[2(R)-Amino-3-mercaptopropyl]-L-3-trans-ethylproline-2,3-dichloro-benzamide N-~2(R)-Amino-3-mercaptopropyl]-D-3-trans-ethylproline-2,3-dichloro-benzamide N-[2(R)-Arnino-3-mercaptopropyl]-L-3-cis-ethylproline-2,3-dichloro-benzamide N-[2(R)-Amino-3-mercaptopropyl]-D-3-cis-ethylproline-2,3-dichloro-benzamide N-[2(R)-Amino-3-mercaptopropyl]-L-3-trans-ethylproline-1-naphthylmethyl amide N-[2(R)-Amino-3-mercaptopropyl]-D-3-trans-ethylproline- 1 -naphthyl-methyl amide WO 9"~3~0 PCT~US95/12284 2~0~3~
N- [2(R)-Amino-3-mercaptopropyl]-L-proline-2,3-dimethylphenyl amide N-[2(R)-Arnino-3-mercaptopropyl]-L-3-trans-ethylproline-2,3-di-5 methylphenyl amide N-[2(R)-Amino-3-mercaptopropyl]-D-3-trans-ethylproline-2,3-dimethylphenyl amide or the pharmaceutically acceptable salts thereof.
The most preferred compounds of the invention are:
N-[2(R)-Amino-3-mercaptopropyl]-L-3-trans-ethylproline- 2,3-dichloroben7.~mide ~
~CI
O~NH Cl L~H
H2N N~\
\
HS
N-[2(R)-Amino-3 -mercaptopropyl] -L-3-cis-ethylproline-2,3-25 dichloroben77~mide ~CI
0~,, NH Cl ~H
H2N N/~ \
\
HS
WO 9''~3~0 PCT/US95/12284 2~0I347 . -N-[2(R)-Amino-3-mercaptopropyl]-L-3-trans-ethylproline-1-naphthylmethyl amide :~
O~,NHH
HS
N-[2(R)-Amino-3-mercaptopropyl] -L-3 -trans-ethylproline-2,3-dimethylphenyl amide l~q~CH3 ~CH3 O~NHH
H2N N~\
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:
3 o Alanine Ala A
Arginine Arg R
Asparagine Asn N
Asparticacid Asp D
Asparagine or WO ~6103810 PCTIUS~5/12284 2~,01347 - ' ' ' Asparticacid Asx Cysteine Cys C
Glllt~mine Gln Q
Glutamic acid Glu E
s Gl~lt~mine or Glutamicacid Glx Z
Glycine Gly G
Histidine His H
Isoleucine ne Leucine Leu L
Lysine Lys K
Methionine Met M
Phenyl~l~nine Phe F
Proline Pro P
Serine Ser S
Threonine Thr T
Tryptophan TIP 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 2s isomers, being included in the present invention.
As used herein, "alkyl" is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms.
As used herein, "cycloalkyl" is intended to include non-3 0 aromatic cyclic hydrocarbon groups having the specified number of carbon atoms. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like.
"Alkenyl" groups include those groups having the specified number of carbon atoms and having one or several double WO 3~ 20 PCT/US95/12284 , ~
2~Q.1~47~
bonds. Examples of alkenyl groups include vinyl, allyl, isopropenyl, pentenyl, hexenyl, heptenyl, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, 1-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 include phenyl, naphthyl, anthracenyl, biphenyl, tetrahydronaphthyl, indanyl, phenanthrenyl and the like.
o The term heterocycle or heterocyclic, as used herein, represents a stable 5- to 7-membered monocyclic or stable 8- to 11-membered bicyclic or stable 11-15 membered tricyclic heterocycle ring which is either saturated or unsaturated, and which consists of carbon atoms and from one to four heteroatoms selected from the 15 group consisting of N, O, and S, and including any bicyclic group in which any of the above-defined heterocyclic rings is fused to a benzene ring. The heterocyclic ring may be attached at any heteroatom or carbon atom which results in the creation of a stable structure. Examples of such heterocyclic elements include, but are not 20 limited to, azepinyl, benzimidazolyl, benzisoxazolyl, benzofurazanyl, benzopyranyl, benzothiopyranyl, benzofuryl, benzothiazolyl, benzothienyl, benzoxazolylj chromanyl, cinnolinyl, dihydrobenzofuryl, dihydro-benzothienyl, dihydrobenzothiopyranyl, dihydrobenzothio-pyranyl sulfone, furyl, imidazolidinyl, imidazolinyl, 25 imidazolyl, indolinyl, indolyl, isochromanyl, isoindolinyl, isoquinolinyl, isothiazolidinyl, isothiazolyl, isothiazolidinyl, morpholinyl, naphthyridinyl, ox~ 7.olyl, 2-oxoazepinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, piperidyl, piperazinyl, pyridyl, pyridyl N-oxide, pyridonyl, pyrazinyl, 30 pyrazolidinyl, pyrazolyl, pyrimi~linyl, pyrrolidinyl, pyrrolyl, quinazolinyl, quinolinyl, quinolinyl N-oxide, quinoxalinyl, tetrahydrofuryl, tetrahydroisoquinolinyl, tetrahydro-quinolinyl, thiamorpholinyl, thiamorpholinyl sulfoxide, thiazolyl, thiazolinyl, thienofuryl, thienothienyl, and thienyl.
2~013~1 As used herein, the terms "substituted aryl", "substituted heterocycle" and "substituted cycloaL~yl" 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, NH2, N(Cl-C6 alkyl)2, N02, (Cl-C6 alkyl)O-, -OH, (Cl-C6 alkyl)S(O)m-, (Cl-C6 alkyl)C(O)NH-, CN, H2N-c(NH)-~ (cl-c6 alkyl)C(O)-, (Cl-C6 alkyl)OC(O)-, N3,(Cl-C6 alkyl)OC(O)NH- and Cl-C20 alkyl.
The following structure:
~N~
~3H2)t represents a cyclic amine moiety having 5 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:
N ~ N ~ N ~,' N ~' It is also understood that substitution on the cyclic amine moiety by 3 0 R2a and R2b may be on different carbon atoms or on the same carbon atom.
The ph~ ceutically acceptable salts of the compounds of this invention include the conventional non-toxic salts of the compounds of this invention as formed, e.g., from non-toxic inorganic WO 9~ 3~20 PCT/US95/12284 ~ 2201347 or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like: and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenyl-acetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxy-benzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, trifluoroacetic and the like.
0 It is intended that the definition of any substituent or variable (e.g., R5, etc.) at a particular location in a molecule be independent of its definitions elsewhere in that molecule. Thus, -N(R5)2 represents -NHH, -NHCH3, -NHC2H5, etc. It is understood that substituents and substitution patterns on the compounds of the instant invention can be selected by one of ordinary skill in the art to provide compounds that are chemically stable and that can be readily synthesized by techniques known in the art as well as those methods set forth below.
The ph~ ceutically acceptable salts of the compounds 2 o of this invention can be synthesized from the compounds of this invention which contain a basic moiety by conventional chemical methods. Generally, the salts are prepared by reacting the free base with stoichiometric amounts or with an excess of the desired salt-forming inorganic or organic acid in a suitable solvent or various combinations of solvents.
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 methods of peptide synthesis are disclosed, for example, in the following works: Schroeder et al., "The Peptides", Vol. I, Academic Press 1965, or Bo~l~n~7ky et al., "Peptide Synthesis", Interscience Publishers, 1966, or McOmie (ed.) "Protective Groups in Organic Chemistry", Plenum Press, 1973, or Barany et al., "The Peptides:
Analysis, Synthesis, Biology" 2, Chapter 1, Academic Press, 1980, or WO 96/09820 PCT/USg5/12284 22013~7 Stewart et al., "Solid Phase Peptide Synthesis", Second Edition, Pierce Chemical Company, 1984. The teachings of these works are hereby incorporated by reference.
Abbreviations used in the description of the chemistry 5 and in the Examples that follow are:
Ac2O Acetic anhydride;
Boc t-Butoxycarbonyl;
DBIJ 1,8-diazabicyclo[5.4.0]undec-7-ene;
DMAP 4-Dimethylaminopyridine;
DME 1,2-Dimethoxyethane;
DMF Dimethylform~mide;
EDC 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide-hydrochloride;
HOBT l-Hydroxybenzotriazole hydrate;
Et3N Triethylamine;
EtOAc Ethyl acetate;
FAB Fast atom bombardment;
HOOBT 3-Hydroxy-1,2,2-benzotriazin-4(3H)-one;
HPLC High-performance liquid chromatography;
MCPBA m-Chloroperoxybenzoic acid;
MsCl Methanesulfonyl 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-F, in 3 0 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.
WO ~-J~g~20 PCT/US95/12284 ~ ~2Q1347 REACTION SCHEME A
O O
>~O~ ~OH EDC, HOBTor HOOBt + HNR3R4a O O
>~ NR3R4aHCI or TFA
( ~H2)t O STrityl H ~NR3R4a >~oJ~H
~P~ H O
(~H2)t ~ J NaCNBH3 NR3R4a TFA
/ ~ (CjH2)t Et3SiH
2s TritylS /
~ NR3R4a / (~ ((~H2)t HS /
WO ~G~S20 PCTIUS95112284 REACTION SCHEME B.
O~N H
>,O~N ~J~X
TritylS / C~,9H2)t Saponify (X _ ester), then \ J~ 1. EDC, HOBT, NHR3 - ~ NHR3 2. TFA, Et3SiH
HS ( (9H2h /
where X is OH or an ester.
In Reaction Scheme A, coupling of a protected amino acid with the a~ropliate amine is accomplished using standard peptide coupling conditions. The resulting amino acid amide is deprotected and the primary amine is reductively alkylated with a protected cysteine-derived aldehyde using sodium cyanoborohydride or sodium triacetoxyborohydride. Finally, removal of the protecting groups provides the compounds of interest.
In Reaction Scheme B, a different strategy for synthesis is described. Reductive alkylation of an amino acid provides a protected dipeptide isostere. The same versatile intermediate can be obtained by reductive alkylation of an amino acid ester followed by saponification. This intermediate can be coupled with any of a :
WO ~'J~`9~20 PCT/US95/12284 .
22~1 3~7 number of amines using standard peptide coupling conditions.
Deprotection provides the active farnesyl transferase inhibitors.
The choice of protecting groups shown in the scheme is not unique and the chemical reactions employed in these syntheses are 5 compatible with other amine and sulfur protecting groups commonly used in peptide synthesis.
Certain compounds of this invention wherein X-Y is an ethenylene or ethylene unit are prepared by employing the reaction sequences shown in Reaction Schemes C and D. Reaction Scheme C
o outlines the preparation of the alkene isosteres lltili7ing standard manipulations such as Wittig reaction, peptide coupling reaction, reductive alkylation, etc., as may be known in the literature or exemplified in the Experimental Procedure. For simplicity, substituents R2a and R2b on the cyclic amine moiety are not shown.
15 It is, however, understood that the reactions illustrated are also applicable to a~ropriately substituted cyclic amine compounds. In Step B of Scheme C, the cysteinyl amino terminlls sidechain, designated Rx is incorporated using coupling reaction A and proRXCOOH; or the alkylation reaction C using proRXCHO and a 20 reducing agent. The Rx sidechain is exposed by deprotection of the sulfur moiety.
The alkane analogs are prepared in a similar manner by including an additional catalytic hydrogenation step as outlined in Reaction Scheme D.
WO ~GI'~820 PCT/IJS95112284 , 22~1347 - 22 -REACTION SCHEME C
~H Ph3P=CHR3 Boc R3 C~H2,t Step A C~H2)t 1. HCI
o 2. NaCNBH3 RXCH2 proRXCHO f ~ ~, R3 3. TFA/Et3SiH ~ ,~H2)t Step B
wherein HS Ph3CS
RX= 3--~ proRX=
R1HN R1HN s5 WO ~IO~B20 2~13 4 7 PCT/US95/12284 REACTION SCHEME C (CONT'D) or Alternate Step B
1. HCI
1l 2. proRXCOH 3. TFA/Et3SiH
EDC, HOBT
o RX ~
f~
~H2)t -WO ~6/~9~20 PCI/USg5/12284 22~ 13~ - 24 -REACTION SCHEME D
~ ~,R3 H2, 5% Pd/C Boc R3 ~H2)t Step A (~H2)t 1. HCI
2. NaCNBH3 RXCH2 proRXCHO ~ ~ R3 3. TFA/Et3SiH ( (~H2)t Step B
or Alternate Step B
1. HCI RX l~
~ ~ R3 Il ~ (5~H2)t 2. proRXCOH
EDC, HOBT
3. TFA/Et3SiH
WO ~GI~5~20 PCT/US95/12284 ~ 25 ~ ~ . r The oxa isostere compounds of this invention are prepared according to the routes outlined in Schemes E and F.
Referring to Scheme E, an aminoalcohol 1 is persilylated with 5 trimethylsilyl chloride and then selectively desilylated with limited methanol to yield amine 2. The nitrogen of the amine 2 is then protected and the alcohol unblocked with excess aqueous methanol to provide 3. Alkylation of 3 with R3XL, where XL is a leaving group such as Br-, I- or Cl- in the presence of a suitable base, preferably NaH, affords 4. Deprotection of alkylated compound 4 provides 5, which undergoes reductive alkylation in the presence of an aldehyde proRXCHO (6) and a reducing agent (e.g., sodium cyanoboro-hydride); or acylation in the presence of proRXCOOH (7) and a peptide coupling reagent affording, after deprotection of the 15 sulfhydryl moiety, the products 8 and 2.
An alternative method for the preparation of the prolyl oxa isostere (compounds 8 and 2 ) is illustrated in Scheme F.
Referring to Scheme F, the aminoalcohol 1 is protected with trifluoroacetic anhydride and the blocked compound 10 treated with 20 diphenyl disulfide in the presence of tributylphosphine to provide the thioether 11. Chlorination of compound 11 provides compound 12 which can be reacted with a variety of alcohols, R30H, in the presence of silver perchlorate and tin (II) chloride, to afford the mixed acetal 4. Removal of the phenylmercapto moiety with Raney nickel 25 followed by deprotection of alkylated compound 14 provides the free amine intermediate, which undergoes reductive alkylation in the presence of an aldehyde proRXCHO (6) and a reducing agent (e.g., sodium cyanoboro-hydride); or acylation in the presence of proRXCOOH (7) and a peptide coupling reagent affording, after 3 0 deprotection of the sulfhydryl moiety, the products 8 and 2.
Yet another alternative method for the preparation of the prolyl oxa isostere (compounds 8 and 2 ) is described in the literature [Ruth E. TenBrink, J. Org. Chem., 52:418-422 (1987)].
WO ~G/0~820 PCT/US95112284 2201~ 26-SCHEME E
OH OSiMe3 ~OH
1 excess Zl~H2)t 2. excess (~
MeOH aq. MeOH
oR3 oR3 f HCI
R3X; (~H2)t MeOH ~CH2h 1. proRXCHO, NaCNBH3 RXCH2 6 ~--o~R3 2. TFA/Et3SiH ~H2)t 1. proRXCOOH, EDC, HOBT
7 Rx l~
2. TFAlEt3SiH ~l--o~ R3 ~H2)t WO 9GI'~820 PCT/US95112284 ~ 2201347 SCHEME F
~OH CF3C)2 CF 8C ~ Ph-S-S-Ph H~H2)~ CH2)t n~U3P
O SPh N-chloro- o Cl SPh Il ~ succinimide ll ~ ~
CF3C~5H2~t 9 4 , CF3C ~ ~ Raney N
SnCI2 ~/~ (C~ H2)t 4A Mol. sieves J
WO 9GI~,~X20 PCT/US95/12284 220 13 ~7 28 -SCHEME F (CON~'D) 1l 2. proRXCHO, NaCNBH3 RXCH2 CF3C 3. TFA/Et3SiH
(~--1. aq.HCI ( ~;~,R3 14 2. proRXCOOH, EDC, HOBT o 3. TFA/Et3SiH \ o~ R3 2)t The thia, oxothia and dioxothia isostere compounds of this invention are prepared in accordance to the route depicted In Scheme G. Aminoalcohol 1 is derivatized with trifluoroacetic anhydride to give 9. Mesylation of 9 provided 15 which is reacted 20 with an appropriate mercaptan to provide 16. The sulfide 16 is readily oxidized to either the sulfoxide or the sulfone by the use of MCPBA
~m-chloroperoxybenzoic acid). Removal of the triflate group in 16 with aqueous HCl the amine 17. This amine hydrochloride 17 undergoes reductive alkylation in the presence of an aldehyde RXCHO
25 (6) and a reducing agent (e.g., sodium cyanoborohydride); or acylation in the presence of RXCOOH (7) and a peptide coupling reagent to afford, after deprotection of the sulfhydryl moiety, the products 18 and 19.
~ 22013~7 SCHEME G
OH O
1l O ~OH
J" CF3C)2O ll ¦ MsCI
H~H2)t ~CH2)t Et N
o~OMs o SR3 ll R3SH ll ~ HCI
CF3C ~, (CH2)t , CF3C ~ , ( H2)t .
H 1. proRXCHO, NaCNBH3 C~;s~R3 2.TFAEt3SiH C~H~)t 1. proRXCOOH, EDC, HOBT
2. TFA/Et3SiH . --S' R3 H2)t ~ 19 WO ~GI'~ 20 PCTIUS95/12284 22013~7 The compounds of this invention inhibit Ras farnesyl transferase which catalyzes the first step in the post-translational processing of Ras and the biosynthesis of functional Ras protein.
These compounds are useful as ph~rm~ceutical agents for m~mrn~lc, especially for hllm~n~. These compounds may be ~lmini~tered to patients for use in the treatment of cancer. Examples of the type of cancer which may be treated with the compounds of this invention include, but are not limited to, colorectal carcinoma, exocrine pancreatic carcinoma, and myeloid leukemias.
The compounds of this invention may be ~lmini~tered to m~mm~l~, preferably hllm~n~, either alone or, preferably, in combination with ph~rm~eutically acceptable carriers or diluents, optionally with known adjuvants, such as alum, in a ph~rm~ceutical composition, according to standard ph~rm~ceutical practice. The compounds can be ~lmini.~tered orally or parenterally, including the intravenous, intramuscular, intraperitoneal, subcutaneous, rectal and topical routes of administration.
For oral use of a chemotherapeutic compound according to this invention, the selected compound may be ~lmini~tered, for example, in the form of tablets or capsules, or as an aqueous solution or suspension. In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch, and lubricating agents, such as magnesium stearate, are commonly added. For oral ~lmini~tration 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 ~he preparation isotonic.
WO ~6/03~20 PCTIUS95/12284 .... s The present invention also encompasses a ph~ ceutical composition useful in the treatment of cancer, comprising the ~lmini~tration of a therapeutically effective amount of the compounds of this invention, with or without ph~ ceutically 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.
o When a compound according to this invention is a-lmini~tered into a hllm~n 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 ~lmini~tered to a m~mm~l undergoing treatment for cancer. Atlmini~tration occurs in an amount between about 0.1 mg~g of body weight to about 20 mg/kg of body weight per day, preferably of between 0.5 mg/kg of body weight to about 10 mg/l~g of body weight per day.
The compounds of the instant invention are also useful as a component in an assay to rapidly determine the presence and quantity of farnesyl-protein transferase (FPTase) in a composition.
Thus the composition to be tested may be divided and the two portions contacted with ~ lules which comprise a known substrate of FPTase (for example a tetrapeptide having a cysteine at the amine te~ s) 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 3 0 allow the FPTase to farnesylate the substrate, the chemical content of the assay mixtures may be determined by well known immunological, radiochemical or chromatographic techniques. Because the compounds of the instant invention are selective inhibitors of FPTase, absence or qll~ntit~tive reduction of the amount of substrate in the Wo ~GI'~,3~20 PCT/US~5112284 22013~7 assay mixture without the 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 tissue samples wkich 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 aliquo~s of a tissue extract cont~ining an unknown amount of farnesyl-protein transferase, an excess amount of a known substrate of FPTase (for example a tetrapeptide having a cysteine at the amine terminus) and farnesyl pyrophosphate are incubated for an appropriate period of time in the presence of varying concentrations of a compound of the instant invention. The concentration of a sufficiently potent inhibitor (i.e., one that has a Ki subst~nti~lly 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.
EXAMPLES
Examples provided are intended to assist in a further understandingoftheinvention. Particularmaterialsemployed, species and conditions are intended to be further illustrative of the invention and not limit~tive 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 over sodium sulfate and evaporated in vacuo on a rotary evaporator.
-WO 9f'~20 PCT/US9S/12284 ~Z~ 7 . .
N-r2(R)-Amino-3-mercaptopropyll-L-proline-2.3-dichlorobenzamide Step A: 2(R)-t-Butoxycarbonylamino-3-triphenylmethyl-mercaptopropanal The aldehyde was synthesized by the method described in U.S. Pat. No. 5,238,922 (Col. 10).
H NMR (CDCl3) o 9.64 (s, lH), 7.42 (m, 6H), 7.28 (m, 9H), 2.45 (m, 2H), 2.38 (m, 2H) Step B: [2(R)-(t-butyloxycarbonyl)amino-3-triphenylmethyl-mercaptopropyll-L-proline methyl ester Proline methyl ester hydrochloride (0.166 g, 1 mmol) and 2-lS t-butoxycarbonylamino-3-triphenylmethylmercaptopropanal (0.538 g, 1.2 mmol) were dissolved in MeOH (8 mT ), treated with 3A molecular sieves (0.16 g), KOAc (0.98 g, 1 mmol) and solid sodium cyanoborohydride (0.094 g, 1.5 mrnol), then stirred at ambient temperature for 24 hr. The reaction mixture was filtered, concentrated, then the residue was partitioned between EtOAc and aq satd NaHCO3 soln. The organic layer was washed with brine, dried (Na2SO4), filtered and concentrated to provide the title compound after chromatography (sio2) with EtOAc: hexane, 1:5. lH NMR (CDCl3) ~7.2-7.4 (m, l5H),
~ 2201347 C ~
t ' ., ;-, -R2a and R2b are independently selected from:
a) hydrogen, b) Cl-C6 alkyl unsubstituted or substituted by aryl, heterocycle, cycloalkyl, alkenyl, R50-, R5S(o)m-, R5C(o)NR5-, CN, (R5)2N-C(NR5)-, R5C(o)-, R50C(o)-, N3, -N(R5)2, or R6oC(o)NR5-, and c) aryl, heterocycle, cycloalkyl, alkenyl, RS0-, R5S(o)m-~ R5C(o)NR5-, CN, N02, (R5)2N-C(NR5)-, R5C(o)-, R50C(o)-, N3, -N(R5)2, or R60C(O)NRS-, R3 is selected from:
a) unsubstituted or substituted aryl, b) unsubstituted or substituted heterocycle, c) unsubstituted or substituted cycloalkyl, and d) C1-C6 alkyl substituted with an unsubstituted or substituted group selected from aryl, heterocycle and cycloalkyl;
X-Y is R4a a) ~sss~Nsss . or o R4b b)\~s~N~ss R4a is selected from a) hydrogen, b) unsubstituted or substituted aryl, c) unsubstituted or substituted heterocycle, d) unsubstituted or substituted cycloaL~yl, and WO 9CI'u5~10 PCT/US95/12284 22013~7 e) C 1 -C6 aL~yl substituted with hydrogen or an unsubstituted or substituted group selected from aryl, heterocycle and cycloalkyl;
wherein heterocycle is selected from pyrrolidinyl, imidazolyl, pyridinyl, thiazolyl, pyridonyl, 2-oxopiperidinyl, indolyl, quinolinyl, isoquinolinyl, and thienyl;
R4b is selected from o a) hydrogen, b) unsubstituted or substituted aryl, c) unsubstituted or substituted heterocycle, d) unsubstituted or substituted cycloaL~yl, e) C 1 -C6 aL~yl substituted with hydrogen or an unsubstituted or substituted group selected from aryl, heterocycle and cycloalkyl, f) a carbonyl group which is bonded to an unsubstituted or substituted group selected from aryl, heterocycle, cycloalkyl and Cl -C6 alkyl substituted with hydrogen or an unsubstituted or substituted group selected from aryl, heterocycle and cycloalkyl, and g) a sulfonyl group which is bonded to an unsubstituted or substituted group selected from aryl, heterocycle, cycloaL~yl and Cl -C6 alkyl substituted with hydrogen or an unsubstituted or substituted group selected from aryl, heterocycle and cycloalkyl;
wherein heterocycle is selected from pyrrolidinyl, imidazolyl, pyridinyl, thiazolyl, pyridonyl, 2 oxopiperidinyl, indolyl, quinolinyl, isoquinolinyl, and thienyl;
R5 is independently selected from hydrogen, Cl-C6 alkyl and aryl;
R6 is independently selected from Cl-C6 alkyl and aryl;
WO~GI'~ O PCT/US95112284 22013~7 Z is independently H2 or 0;
m is 0, 1 or 2, provided that m is 0 when R5 = hydrogen;
nis 0,1,2,30r4;and t is 3, 4 or 5;
or the pharmaceutically acceptable salts thereof.
The preferred compounds of this invention are as follows:
N-[2(R)-Amino-3 -mercaptopropyl] -L-proline-2,3 -dichlorobenzylamide N-[2(R)-Amino-3-mercaptopropyl]-L-proline-1-naphthylmethyl amide N-[2(R)-Amino-3-mercaptopropyl] -L-pipecolyl-2,3-dichlorobenzamide N-[2(R)-Amino-3-mercaptopropyl]-L-3-trans-ethylproline-2,3-dichloro-benzamide N-~2(R)-Amino-3-mercaptopropyl]-D-3-trans-ethylproline-2,3-dichloro-benzamide N-[2(R)-Arnino-3-mercaptopropyl]-L-3-cis-ethylproline-2,3-dichloro-benzamide N-[2(R)-Amino-3-mercaptopropyl]-D-3-cis-ethylproline-2,3-dichloro-benzamide N-[2(R)-Amino-3-mercaptopropyl]-L-3-trans-ethylproline-1-naphthylmethyl amide N-[2(R)-Amino-3-mercaptopropyl]-D-3-trans-ethylproline- 1 -naphthyl-methyl amide WO 9"~3~0 PCT~US95/12284 2~0~3~
N- [2(R)-Amino-3-mercaptopropyl]-L-proline-2,3-dimethylphenyl amide N-[2(R)-Arnino-3-mercaptopropyl]-L-3-trans-ethylproline-2,3-di-5 methylphenyl amide N-[2(R)-Amino-3-mercaptopropyl]-D-3-trans-ethylproline-2,3-dimethylphenyl amide or the pharmaceutically acceptable salts thereof.
The most preferred compounds of the invention are:
N-[2(R)-Amino-3-mercaptopropyl]-L-3-trans-ethylproline- 2,3-dichloroben7.~mide ~
~CI
O~NH Cl L~H
H2N N~\
\
HS
N-[2(R)-Amino-3 -mercaptopropyl] -L-3-cis-ethylproline-2,3-25 dichloroben77~mide ~CI
0~,, NH Cl ~H
H2N N/~ \
\
HS
WO 9''~3~0 PCT/US95/12284 2~0I347 . -N-[2(R)-Amino-3-mercaptopropyl]-L-3-trans-ethylproline-1-naphthylmethyl amide :~
O~,NHH
HS
N-[2(R)-Amino-3-mercaptopropyl] -L-3 -trans-ethylproline-2,3-dimethylphenyl amide l~q~CH3 ~CH3 O~NHH
H2N N~\
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:
3 o Alanine Ala A
Arginine Arg R
Asparagine Asn N
Asparticacid Asp D
Asparagine or WO ~6103810 PCTIUS~5/12284 2~,01347 - ' ' ' Asparticacid Asx Cysteine Cys C
Glllt~mine Gln Q
Glutamic acid Glu E
s Gl~lt~mine or Glutamicacid Glx Z
Glycine Gly G
Histidine His H
Isoleucine ne Leucine Leu L
Lysine Lys K
Methionine Met M
Phenyl~l~nine Phe F
Proline Pro P
Serine Ser S
Threonine Thr T
Tryptophan TIP 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 2s isomers, being included in the present invention.
As used herein, "alkyl" is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms.
As used herein, "cycloalkyl" is intended to include non-3 0 aromatic cyclic hydrocarbon groups having the specified number of carbon atoms. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like.
"Alkenyl" groups include those groups having the specified number of carbon atoms and having one or several double WO 3~ 20 PCT/US95/12284 , ~
2~Q.1~47~
bonds. Examples of alkenyl groups include vinyl, allyl, isopropenyl, pentenyl, hexenyl, heptenyl, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, 1-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 include phenyl, naphthyl, anthracenyl, biphenyl, tetrahydronaphthyl, indanyl, phenanthrenyl and the like.
o The term heterocycle or heterocyclic, as used herein, represents a stable 5- to 7-membered monocyclic or stable 8- to 11-membered bicyclic or stable 11-15 membered tricyclic heterocycle ring which is either saturated or unsaturated, and which consists of carbon atoms and from one to four heteroatoms selected from the 15 group consisting of N, O, and S, and including any bicyclic group in which any of the above-defined heterocyclic rings is fused to a benzene ring. The heterocyclic ring may be attached at any heteroatom or carbon atom which results in the creation of a stable structure. Examples of such heterocyclic elements include, but are not 20 limited to, azepinyl, benzimidazolyl, benzisoxazolyl, benzofurazanyl, benzopyranyl, benzothiopyranyl, benzofuryl, benzothiazolyl, benzothienyl, benzoxazolylj chromanyl, cinnolinyl, dihydrobenzofuryl, dihydro-benzothienyl, dihydrobenzothiopyranyl, dihydrobenzothio-pyranyl sulfone, furyl, imidazolidinyl, imidazolinyl, 25 imidazolyl, indolinyl, indolyl, isochromanyl, isoindolinyl, isoquinolinyl, isothiazolidinyl, isothiazolyl, isothiazolidinyl, morpholinyl, naphthyridinyl, ox~ 7.olyl, 2-oxoazepinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, piperidyl, piperazinyl, pyridyl, pyridyl N-oxide, pyridonyl, pyrazinyl, 30 pyrazolidinyl, pyrazolyl, pyrimi~linyl, pyrrolidinyl, pyrrolyl, quinazolinyl, quinolinyl, quinolinyl N-oxide, quinoxalinyl, tetrahydrofuryl, tetrahydroisoquinolinyl, tetrahydro-quinolinyl, thiamorpholinyl, thiamorpholinyl sulfoxide, thiazolyl, thiazolinyl, thienofuryl, thienothienyl, and thienyl.
2~013~1 As used herein, the terms "substituted aryl", "substituted heterocycle" and "substituted cycloaL~yl" 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, NH2, N(Cl-C6 alkyl)2, N02, (Cl-C6 alkyl)O-, -OH, (Cl-C6 alkyl)S(O)m-, (Cl-C6 alkyl)C(O)NH-, CN, H2N-c(NH)-~ (cl-c6 alkyl)C(O)-, (Cl-C6 alkyl)OC(O)-, N3,(Cl-C6 alkyl)OC(O)NH- and Cl-C20 alkyl.
The following structure:
~N~
~3H2)t represents a cyclic amine moiety having 5 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:
N ~ N ~ N ~,' N ~' It is also understood that substitution on the cyclic amine moiety by 3 0 R2a and R2b may be on different carbon atoms or on the same carbon atom.
The ph~ ceutically acceptable salts of the compounds of this invention include the conventional non-toxic salts of the compounds of this invention as formed, e.g., from non-toxic inorganic WO 9~ 3~20 PCT/US95/12284 ~ 2201347 or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like: and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenyl-acetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxy-benzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, trifluoroacetic and the like.
0 It is intended that the definition of any substituent or variable (e.g., R5, etc.) at a particular location in a molecule be independent of its definitions elsewhere in that molecule. Thus, -N(R5)2 represents -NHH, -NHCH3, -NHC2H5, etc. It is understood that substituents and substitution patterns on the compounds of the instant invention can be selected by one of ordinary skill in the art to provide compounds that are chemically stable and that can be readily synthesized by techniques known in the art as well as those methods set forth below.
The ph~ ceutically acceptable salts of the compounds 2 o of this invention can be synthesized from the compounds of this invention which contain a basic moiety by conventional chemical methods. Generally, the salts are prepared by reacting the free base with stoichiometric amounts or with an excess of the desired salt-forming inorganic or organic acid in a suitable solvent or various combinations of solvents.
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 methods of peptide synthesis are disclosed, for example, in the following works: Schroeder et al., "The Peptides", Vol. I, Academic Press 1965, or Bo~l~n~7ky et al., "Peptide Synthesis", Interscience Publishers, 1966, or McOmie (ed.) "Protective Groups in Organic Chemistry", Plenum Press, 1973, or Barany et al., "The Peptides:
Analysis, Synthesis, Biology" 2, Chapter 1, Academic Press, 1980, or WO 96/09820 PCT/USg5/12284 22013~7 Stewart et al., "Solid Phase Peptide Synthesis", Second Edition, Pierce Chemical Company, 1984. The teachings of these works are hereby incorporated by reference.
Abbreviations used in the description of the chemistry 5 and in the Examples that follow are:
Ac2O Acetic anhydride;
Boc t-Butoxycarbonyl;
DBIJ 1,8-diazabicyclo[5.4.0]undec-7-ene;
DMAP 4-Dimethylaminopyridine;
DME 1,2-Dimethoxyethane;
DMF Dimethylform~mide;
EDC 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide-hydrochloride;
HOBT l-Hydroxybenzotriazole hydrate;
Et3N Triethylamine;
EtOAc Ethyl acetate;
FAB Fast atom bombardment;
HOOBT 3-Hydroxy-1,2,2-benzotriazin-4(3H)-one;
HPLC High-performance liquid chromatography;
MCPBA m-Chloroperoxybenzoic acid;
MsCl Methanesulfonyl 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-F, in 3 0 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.
WO ~-J~g~20 PCT/US95/12284 ~ ~2Q1347 REACTION SCHEME A
O O
>~O~ ~OH EDC, HOBTor HOOBt + HNR3R4a O O
>~ NR3R4aHCI or TFA
( ~H2)t O STrityl H ~NR3R4a >~oJ~H
~P~ H O
(~H2)t ~ J NaCNBH3 NR3R4a TFA
/ ~ (CjH2)t Et3SiH
2s TritylS /
~ NR3R4a / (~ ((~H2)t HS /
WO ~G~S20 PCTIUS95112284 REACTION SCHEME B.
O~N H
>,O~N ~J~X
TritylS / C~,9H2)t Saponify (X _ ester), then \ J~ 1. EDC, HOBT, NHR3 - ~ NHR3 2. TFA, Et3SiH
HS ( (9H2h /
where X is OH or an ester.
In Reaction Scheme A, coupling of a protected amino acid with the a~ropliate amine is accomplished using standard peptide coupling conditions. The resulting amino acid amide is deprotected and the primary amine is reductively alkylated with a protected cysteine-derived aldehyde using sodium cyanoborohydride or sodium triacetoxyborohydride. Finally, removal of the protecting groups provides the compounds of interest.
In Reaction Scheme B, a different strategy for synthesis is described. Reductive alkylation of an amino acid provides a protected dipeptide isostere. The same versatile intermediate can be obtained by reductive alkylation of an amino acid ester followed by saponification. This intermediate can be coupled with any of a :
WO ~'J~`9~20 PCT/US95/12284 .
22~1 3~7 number of amines using standard peptide coupling conditions.
Deprotection provides the active farnesyl transferase inhibitors.
The choice of protecting groups shown in the scheme is not unique and the chemical reactions employed in these syntheses are 5 compatible with other amine and sulfur protecting groups commonly used in peptide synthesis.
Certain compounds of this invention wherein X-Y is an ethenylene or ethylene unit are prepared by employing the reaction sequences shown in Reaction Schemes C and D. Reaction Scheme C
o outlines the preparation of the alkene isosteres lltili7ing standard manipulations such as Wittig reaction, peptide coupling reaction, reductive alkylation, etc., as may be known in the literature or exemplified in the Experimental Procedure. For simplicity, substituents R2a and R2b on the cyclic amine moiety are not shown.
15 It is, however, understood that the reactions illustrated are also applicable to a~ropriately substituted cyclic amine compounds. In Step B of Scheme C, the cysteinyl amino terminlls sidechain, designated Rx is incorporated using coupling reaction A and proRXCOOH; or the alkylation reaction C using proRXCHO and a 20 reducing agent. The Rx sidechain is exposed by deprotection of the sulfur moiety.
The alkane analogs are prepared in a similar manner by including an additional catalytic hydrogenation step as outlined in Reaction Scheme D.
WO ~GI'~820 PCT/IJS95112284 , 22~1347 - 22 -REACTION SCHEME C
~H Ph3P=CHR3 Boc R3 C~H2,t Step A C~H2)t 1. HCI
o 2. NaCNBH3 RXCH2 proRXCHO f ~ ~, R3 3. TFA/Et3SiH ~ ,~H2)t Step B
wherein HS Ph3CS
RX= 3--~ proRX=
R1HN R1HN s5 WO ~IO~B20 2~13 4 7 PCT/US95/12284 REACTION SCHEME C (CONT'D) or Alternate Step B
1. HCI
1l 2. proRXCOH 3. TFA/Et3SiH
EDC, HOBT
o RX ~
f~
~H2)t -WO ~6/~9~20 PCI/USg5/12284 22~ 13~ - 24 -REACTION SCHEME D
~ ~,R3 H2, 5% Pd/C Boc R3 ~H2)t Step A (~H2)t 1. HCI
2. NaCNBH3 RXCH2 proRXCHO ~ ~ R3 3. TFA/Et3SiH ( (~H2)t Step B
or Alternate Step B
1. HCI RX l~
~ ~ R3 Il ~ (5~H2)t 2. proRXCOH
EDC, HOBT
3. TFA/Et3SiH
WO ~GI~5~20 PCT/US95/12284 ~ 25 ~ ~ . r The oxa isostere compounds of this invention are prepared according to the routes outlined in Schemes E and F.
Referring to Scheme E, an aminoalcohol 1 is persilylated with 5 trimethylsilyl chloride and then selectively desilylated with limited methanol to yield amine 2. The nitrogen of the amine 2 is then protected and the alcohol unblocked with excess aqueous methanol to provide 3. Alkylation of 3 with R3XL, where XL is a leaving group such as Br-, I- or Cl- in the presence of a suitable base, preferably NaH, affords 4. Deprotection of alkylated compound 4 provides 5, which undergoes reductive alkylation in the presence of an aldehyde proRXCHO (6) and a reducing agent (e.g., sodium cyanoboro-hydride); or acylation in the presence of proRXCOOH (7) and a peptide coupling reagent affording, after deprotection of the 15 sulfhydryl moiety, the products 8 and 2.
An alternative method for the preparation of the prolyl oxa isostere (compounds 8 and 2 ) is illustrated in Scheme F.
Referring to Scheme F, the aminoalcohol 1 is protected with trifluoroacetic anhydride and the blocked compound 10 treated with 20 diphenyl disulfide in the presence of tributylphosphine to provide the thioether 11. Chlorination of compound 11 provides compound 12 which can be reacted with a variety of alcohols, R30H, in the presence of silver perchlorate and tin (II) chloride, to afford the mixed acetal 4. Removal of the phenylmercapto moiety with Raney nickel 25 followed by deprotection of alkylated compound 14 provides the free amine intermediate, which undergoes reductive alkylation in the presence of an aldehyde proRXCHO (6) and a reducing agent (e.g., sodium cyanoboro-hydride); or acylation in the presence of proRXCOOH (7) and a peptide coupling reagent affording, after 3 0 deprotection of the sulfhydryl moiety, the products 8 and 2.
Yet another alternative method for the preparation of the prolyl oxa isostere (compounds 8 and 2 ) is described in the literature [Ruth E. TenBrink, J. Org. Chem., 52:418-422 (1987)].
WO ~G/0~820 PCT/US95112284 2201~ 26-SCHEME E
OH OSiMe3 ~OH
1 excess Zl~H2)t 2. excess (~
MeOH aq. MeOH
oR3 oR3 f HCI
R3X; (~H2)t MeOH ~CH2h 1. proRXCHO, NaCNBH3 RXCH2 6 ~--o~R3 2. TFA/Et3SiH ~H2)t 1. proRXCOOH, EDC, HOBT
7 Rx l~
2. TFAlEt3SiH ~l--o~ R3 ~H2)t WO 9GI'~820 PCT/US95112284 ~ 2201347 SCHEME F
~OH CF3C)2 CF 8C ~ Ph-S-S-Ph H~H2)~ CH2)t n~U3P
O SPh N-chloro- o Cl SPh Il ~ succinimide ll ~ ~
CF3C~5H2~t 9 4 , CF3C ~ ~ Raney N
SnCI2 ~/~ (C~ H2)t 4A Mol. sieves J
WO 9GI~,~X20 PCT/US95/12284 220 13 ~7 28 -SCHEME F (CON~'D) 1l 2. proRXCHO, NaCNBH3 RXCH2 CF3C 3. TFA/Et3SiH
(~--1. aq.HCI ( ~;~,R3 14 2. proRXCOOH, EDC, HOBT o 3. TFA/Et3SiH \ o~ R3 2)t The thia, oxothia and dioxothia isostere compounds of this invention are prepared in accordance to the route depicted In Scheme G. Aminoalcohol 1 is derivatized with trifluoroacetic anhydride to give 9. Mesylation of 9 provided 15 which is reacted 20 with an appropriate mercaptan to provide 16. The sulfide 16 is readily oxidized to either the sulfoxide or the sulfone by the use of MCPBA
~m-chloroperoxybenzoic acid). Removal of the triflate group in 16 with aqueous HCl the amine 17. This amine hydrochloride 17 undergoes reductive alkylation in the presence of an aldehyde RXCHO
25 (6) and a reducing agent (e.g., sodium cyanoborohydride); or acylation in the presence of RXCOOH (7) and a peptide coupling reagent to afford, after deprotection of the sulfhydryl moiety, the products 18 and 19.
~ 22013~7 SCHEME G
OH O
1l O ~OH
J" CF3C)2O ll ¦ MsCI
H~H2)t ~CH2)t Et N
o~OMs o SR3 ll R3SH ll ~ HCI
CF3C ~, (CH2)t , CF3C ~ , ( H2)t .
H 1. proRXCHO, NaCNBH3 C~;s~R3 2.TFAEt3SiH C~H~)t 1. proRXCOOH, EDC, HOBT
2. TFA/Et3SiH . --S' R3 H2)t ~ 19 WO ~GI'~ 20 PCTIUS95/12284 22013~7 The compounds of this invention inhibit Ras farnesyl transferase which catalyzes the first step in the post-translational processing of Ras and the biosynthesis of functional Ras protein.
These compounds are useful as ph~rm~ceutical agents for m~mrn~lc, especially for hllm~n~. These compounds may be ~lmini~tered to patients for use in the treatment of cancer. Examples of the type of cancer which may be treated with the compounds of this invention include, but are not limited to, colorectal carcinoma, exocrine pancreatic carcinoma, and myeloid leukemias.
The compounds of this invention may be ~lmini~tered to m~mm~l~, preferably hllm~n~, either alone or, preferably, in combination with ph~rm~eutically acceptable carriers or diluents, optionally with known adjuvants, such as alum, in a ph~rm~ceutical composition, according to standard ph~rm~ceutical practice. The compounds can be ~lmini.~tered orally or parenterally, including the intravenous, intramuscular, intraperitoneal, subcutaneous, rectal and topical routes of administration.
For oral use of a chemotherapeutic compound according to this invention, the selected compound may be ~lmini~tered, for example, in the form of tablets or capsules, or as an aqueous solution or suspension. In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch, and lubricating agents, such as magnesium stearate, are commonly added. For oral ~lmini~tration 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 ~he preparation isotonic.
WO ~6/03~20 PCTIUS95/12284 .... s The present invention also encompasses a ph~ ceutical composition useful in the treatment of cancer, comprising the ~lmini~tration of a therapeutically effective amount of the compounds of this invention, with or without ph~ ceutically 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.
o When a compound according to this invention is a-lmini~tered into a hllm~n 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 ~lmini~tered to a m~mm~l undergoing treatment for cancer. Atlmini~tration occurs in an amount between about 0.1 mg~g of body weight to about 20 mg/kg of body weight per day, preferably of between 0.5 mg/kg of body weight to about 10 mg/l~g of body weight per day.
The compounds of the instant invention are also useful as a component in an assay to rapidly determine the presence and quantity of farnesyl-protein transferase (FPTase) in a composition.
Thus the composition to be tested may be divided and the two portions contacted with ~ lules which comprise a known substrate of FPTase (for example a tetrapeptide having a cysteine at the amine te~ s) 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 3 0 allow the FPTase to farnesylate the substrate, the chemical content of the assay mixtures may be determined by well known immunological, radiochemical or chromatographic techniques. Because the compounds of the instant invention are selective inhibitors of FPTase, absence or qll~ntit~tive reduction of the amount of substrate in the Wo ~GI'~,3~20 PCT/US~5112284 22013~7 assay mixture without the 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 tissue samples wkich 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 aliquo~s of a tissue extract cont~ining an unknown amount of farnesyl-protein transferase, an excess amount of a known substrate of FPTase (for example a tetrapeptide having a cysteine at the amine terminus) and farnesyl pyrophosphate are incubated for an appropriate period of time in the presence of varying concentrations of a compound of the instant invention. The concentration of a sufficiently potent inhibitor (i.e., one that has a Ki subst~nti~lly 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.
EXAMPLES
Examples provided are intended to assist in a further understandingoftheinvention. Particularmaterialsemployed, species and conditions are intended to be further illustrative of the invention and not limit~tive 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 over sodium sulfate and evaporated in vacuo on a rotary evaporator.
-WO 9f'~20 PCT/US9S/12284 ~Z~ 7 . .
N-r2(R)-Amino-3-mercaptopropyll-L-proline-2.3-dichlorobenzamide Step A: 2(R)-t-Butoxycarbonylamino-3-triphenylmethyl-mercaptopropanal The aldehyde was synthesized by the method described in U.S. Pat. No. 5,238,922 (Col. 10).
H NMR (CDCl3) o 9.64 (s, lH), 7.42 (m, 6H), 7.28 (m, 9H), 2.45 (m, 2H), 2.38 (m, 2H) Step B: [2(R)-(t-butyloxycarbonyl)amino-3-triphenylmethyl-mercaptopropyll-L-proline methyl ester Proline methyl ester hydrochloride (0.166 g, 1 mmol) and 2-lS t-butoxycarbonylamino-3-triphenylmethylmercaptopropanal (0.538 g, 1.2 mmol) were dissolved in MeOH (8 mT ), treated with 3A molecular sieves (0.16 g), KOAc (0.98 g, 1 mmol) and solid sodium cyanoborohydride (0.094 g, 1.5 mrnol), then stirred at ambient temperature for 24 hr. The reaction mixture was filtered, concentrated, then the residue was partitioned between EtOAc and aq satd NaHCO3 soln. The organic layer was washed with brine, dried (Na2SO4), filtered and concentrated to provide the title compound after chromatography (sio2) with EtOAc: hexane, 1:5. lH NMR (CDCl3) ~7.2-7.4 (m, l5H),
4.75-4.85 (m, lH), 3.67 (s, 3H), 3.6-3.75 (m, lH), 3.18-3.28 (m, lH), 2.9-25 3 0 (m, lH), 2.35-2.65 (m, 4H), 1.75-2.0 (m, 4H), 1.45 (s, 9H), 1.2-1.4 (m, lH).
Step C: [2(R)-(t-butoxycarbonyl)amino-3-triphenylmethyl-mercaptopropyll -L-proline [2(R)-(t-butyloxycarbonyl)amino-3-triphenylmethyl-mercaptopropyl]-L-proline methyl ester (0.325 g, 0.58 mmol) was WO 96/09820 PCI/US9!!i/12284 22013~7 dissolved in MeOH (12 mL) and lN NaOH solution (2.3 mL, 2.3 mmol) with stirring at ambient temperature. After 24 hrs, the reaction mixture was concentrated to remove MeOH, then dissolved in H2O, neutralized with lN HCl (2.3 mL, 2.3 mmol), and extracted with EtOAc (3 x 10 mL).
Step C: [2(R)-(t-butoxycarbonyl)amino-3-triphenylmethyl-mercaptopropyll -L-proline [2(R)-(t-butyloxycarbonyl)amino-3-triphenylmethyl-mercaptopropyl]-L-proline methyl ester (0.325 g, 0.58 mmol) was WO 96/09820 PCI/US9!!i/12284 22013~7 dissolved in MeOH (12 mL) and lN NaOH solution (2.3 mL, 2.3 mmol) with stirring at ambient temperature. After 24 hrs, the reaction mixture was concentrated to remove MeOH, then dissolved in H2O, neutralized with lN HCl (2.3 mL, 2.3 mmol), and extracted with EtOAc (3 x 10 mL).
5 The organics were combined, washed with brine and dried (Na2SO4).
Filtration and concentration to dryness provided the title compound . lH
NMR (CD30D) ~ 7.2-7.5 (m, 15H), 3.7-3.85 (m, lH), 3.5-3.7 (m, 2H), 2.9-3.15 (m, 3H), 2.2-2.6 (m, 3H), 1.8-2.2 (m, 3H), 1.48 (s, 9H).
Step D: N-[2(R)-t-Butoxycarbonylamino-3-triphenylmethyl-mercaptopropyll -L-proline-2.3-dichlorobenzamide L2(R) -(t-butoxycarbonyl)amino-3 -triphenylmethyl-mercaptopropyl]-L-proline (0.08 g, 0.153 mmol), 1-(3-dimethyl-aminopropyl)-3-ethylcarbodiimide (EDC) (0.035 g, 0.184 mmol), and 1-hydroxybenzotriazole hydrate (HOBT) (0.025 g, 0.184 mmol) were dissolved in DMF (2 mL), treated with 2,3-dichlorobenzyl~mine (0.025 mL, 0.184 mmol), and brought to pH 7 with Et3N (0.026 mL, 0.184 mmol). After stirring at ambient temperature for 18 h the mixture was concentrated to dryness, and the residue was partitioned between EtOAc 20 and H2O, the organic layer separated, washed with aq satd NaHCO3, brine, and dried (Na2SO4). Filtration and concentration provided the title compound. 1H NMR (CD30D) ~ 7.1-7.5 (m, 18H), 4.53 (d,lH, J = 8 Hz), 4.26 (d, lH, J = 8 Hz), 3.64-3.76 (m, lH), 2.96-3.14 (m, 2H), 2.52-2.66 (m, lH), 2.1-2.5 (m, 5H), 1.6-1.9 (m, 3H), 1.45 (s, 9H).
Step E: N-[2(R)-Amino-3-mercaptopropyl]-L-proline- 2,3-dichlorobenzamide N-[2(R)-t-Butoxycarbonylamino-3-triphenylmethyl-mercaptopropyl]-L-proline-2,3-dichlorobenzamide (0.097 g, 0.14 mmol) 30 was dissolved in CH2Cl2 (3 mL), CF3CO2H (1 mL) at ambient temperature, treated with triethylsilane (0.088 mL, 0.55 mmol) and stirred for 1 h. The reaction ~ Lule was triturated with 0.1 % TFA in H2O, filtered, concentrated and chromatographed by RP-HPLC and lyophilized. The residue was dissolved in MeOH (1 mL), treated with WO 9~ 320 PCTIUS95/12284 concd HCl, concentrated and triturated with Et20 to provide the title compound as the bis hydrochloride salt. lH NMR (CD30D) o 7.50 (d, r . lH, J = 6 Hz), 7.27-7.4 (m, 2H), 4.60 (ABq, 2H), 4.25-4.7 (m, lH), 3.3-3.9 (m, 5H), 2.99 (d, 2H, J = 5 Hz), 2.5-2.7 (m, lH), 2.0-2.3 (m, 3H).
Anal. calcd for C15H21N30SCl2-2.75 HCl: C, 38.97; H, 5.18, N, 9.09;
found C, 38.63; H, 5.21; N, 8.86.
Using the methods described in Example 1, but a dir~erelll cyclic amino acid in Step B or a different amine in Step D the following compound were prepared:
N-r2(R)-Amino-3-mercaptopropyll-L-proline-l-naphthylmethyl amide Anal. calcd for ClgH25N3OS-2.1 CF3CO2H-0.1 H20: C, 47.66; H, 4.71; N, 7.19; found C, 47.68; H, ~.57; N, 7.02.
N-r2(R)-Arnino-3-mercaptopropyll-L-pipecolvl-2,3-dichlorobenzamide Anal. calcd for C16H23N30SC12-2.5 CF3CO2H: C, 38.13; H, 3.89, N, 6.35; found C, 38.20; H, 3.65; N, 6.68.
N-[2(R)-Amino-3-mercaptopropyl]-L-3-trans-ethylproline-2,3-dichlorobenzamide MS (M~1) = 390.
25 N-[2(R)-Amino-3-mercaptopropyl]-D-3-trans-ethylproline-2,3-dichlorobenzamide Anal. calcd for C17H25N3OSCl2-2.75 CF3C02H: C, 38.39; H, 3.97; N, 5.97;foundC,38.39;H,3.97;N,6.27.
30 N-[2(R)-Arnino-3-mercaptopropyl]-L-3-cis-ethylproline- 2,3-dichlorobenzamide Anal. calcd for Cl7H25N3oscl2-2.75 CF3co2H: C, 38.39; H, 3.97; N, 5.97;foundC,38.51;H,4.18;N,6.28.
WO ~GI'~20 PCT/US~5112284 22~1347 N-[2(R)-Amino-3-mercaptopropyl] -D-3-cis-ethylproline-2,3 -dichloroben7~mide Anal. calcd for C17H25N3OSCl2-2.5 CF3CO2H: C, 39.12; H, 4.10; N,
Filtration and concentration to dryness provided the title compound . lH
NMR (CD30D) ~ 7.2-7.5 (m, 15H), 3.7-3.85 (m, lH), 3.5-3.7 (m, 2H), 2.9-3.15 (m, 3H), 2.2-2.6 (m, 3H), 1.8-2.2 (m, 3H), 1.48 (s, 9H).
Step D: N-[2(R)-t-Butoxycarbonylamino-3-triphenylmethyl-mercaptopropyll -L-proline-2.3-dichlorobenzamide L2(R) -(t-butoxycarbonyl)amino-3 -triphenylmethyl-mercaptopropyl]-L-proline (0.08 g, 0.153 mmol), 1-(3-dimethyl-aminopropyl)-3-ethylcarbodiimide (EDC) (0.035 g, 0.184 mmol), and 1-hydroxybenzotriazole hydrate (HOBT) (0.025 g, 0.184 mmol) were dissolved in DMF (2 mL), treated with 2,3-dichlorobenzyl~mine (0.025 mL, 0.184 mmol), and brought to pH 7 with Et3N (0.026 mL, 0.184 mmol). After stirring at ambient temperature for 18 h the mixture was concentrated to dryness, and the residue was partitioned between EtOAc 20 and H2O, the organic layer separated, washed with aq satd NaHCO3, brine, and dried (Na2SO4). Filtration and concentration provided the title compound. 1H NMR (CD30D) ~ 7.1-7.5 (m, 18H), 4.53 (d,lH, J = 8 Hz), 4.26 (d, lH, J = 8 Hz), 3.64-3.76 (m, lH), 2.96-3.14 (m, 2H), 2.52-2.66 (m, lH), 2.1-2.5 (m, 5H), 1.6-1.9 (m, 3H), 1.45 (s, 9H).
Step E: N-[2(R)-Amino-3-mercaptopropyl]-L-proline- 2,3-dichlorobenzamide N-[2(R)-t-Butoxycarbonylamino-3-triphenylmethyl-mercaptopropyl]-L-proline-2,3-dichlorobenzamide (0.097 g, 0.14 mmol) 30 was dissolved in CH2Cl2 (3 mL), CF3CO2H (1 mL) at ambient temperature, treated with triethylsilane (0.088 mL, 0.55 mmol) and stirred for 1 h. The reaction ~ Lule was triturated with 0.1 % TFA in H2O, filtered, concentrated and chromatographed by RP-HPLC and lyophilized. The residue was dissolved in MeOH (1 mL), treated with WO 9~ 320 PCTIUS95/12284 concd HCl, concentrated and triturated with Et20 to provide the title compound as the bis hydrochloride salt. lH NMR (CD30D) o 7.50 (d, r . lH, J = 6 Hz), 7.27-7.4 (m, 2H), 4.60 (ABq, 2H), 4.25-4.7 (m, lH), 3.3-3.9 (m, 5H), 2.99 (d, 2H, J = 5 Hz), 2.5-2.7 (m, lH), 2.0-2.3 (m, 3H).
Anal. calcd for C15H21N30SCl2-2.75 HCl: C, 38.97; H, 5.18, N, 9.09;
found C, 38.63; H, 5.21; N, 8.86.
Using the methods described in Example 1, but a dir~erelll cyclic amino acid in Step B or a different amine in Step D the following compound were prepared:
N-r2(R)-Amino-3-mercaptopropyll-L-proline-l-naphthylmethyl amide Anal. calcd for ClgH25N3OS-2.1 CF3CO2H-0.1 H20: C, 47.66; H, 4.71; N, 7.19; found C, 47.68; H, ~.57; N, 7.02.
N-r2(R)-Arnino-3-mercaptopropyll-L-pipecolvl-2,3-dichlorobenzamide Anal. calcd for C16H23N30SC12-2.5 CF3CO2H: C, 38.13; H, 3.89, N, 6.35; found C, 38.20; H, 3.65; N, 6.68.
N-[2(R)-Amino-3-mercaptopropyl]-L-3-trans-ethylproline-2,3-dichlorobenzamide MS (M~1) = 390.
25 N-[2(R)-Amino-3-mercaptopropyl]-D-3-trans-ethylproline-2,3-dichlorobenzamide Anal. calcd for C17H25N3OSCl2-2.75 CF3C02H: C, 38.39; H, 3.97; N, 5.97;foundC,38.39;H,3.97;N,6.27.
30 N-[2(R)-Arnino-3-mercaptopropyl]-L-3-cis-ethylproline- 2,3-dichlorobenzamide Anal. calcd for Cl7H25N3oscl2-2.75 CF3co2H: C, 38.39; H, 3.97; N, 5.97;foundC,38.51;H,4.18;N,6.28.
WO ~GI'~20 PCT/US~5112284 22~1347 N-[2(R)-Amino-3-mercaptopropyl] -D-3-cis-ethylproline-2,3 -dichloroben7~mide Anal. calcd for C17H25N3OSCl2-2.5 CF3CO2H: C, 39.12; H, 4.10; N,
6.22; found C, 39.03; H, 4.22; N, 6.47.
N-[2(R)-Amino-3-mercaptopropyl]-L-3-trans-ethylproline- 1 -naphthyl-methyl amide Anal. calcd for C21H29N30S-2.25 CF3co2H: C, 48.76; H, 5.02; N, 6.69; found C, 48.63; H, 5.10; N, 6.89.
N-[2(R)-Amino-3-mercaptopropyl]-D-3-trans-ethylproline- 1 -naphthylmethvl amide Anal. calcd for C21H29N3OS-1.85 CF3CO2H: C, 50.93; H, 5.34; N,
N-[2(R)-Amino-3-mercaptopropyl]-L-3-trans-ethylproline- 1 -naphthyl-methyl amide Anal. calcd for C21H29N30S-2.25 CF3co2H: C, 48.76; H, 5.02; N, 6.69; found C, 48.63; H, 5.10; N, 6.89.
N-[2(R)-Amino-3-mercaptopropyl]-D-3-trans-ethylproline- 1 -naphthylmethvl amide Anal. calcd for C21H29N3OS-1.85 CF3CO2H: C, 50.93; H, 5.34; N,
7.21; found C, 50.88; H, 5.43; N, 7.41.
N-r2(R)-Arnino-3-merca~to~lo~yll-L-prolirle-23-dimethylphenvl amide 20 Step A: N-r2(R)-t-butoxycarbonylamino-3-triphenyl methyl-mercaptopropvll-L-proline-2,3-dimethvlphenyl amide To a solution of 2,3-dimethylaniline (0.05 mL, 0.41 mmol) and diisopropylethylamine (0.12 mL, 0.69 mmol) in CH2Cl2 (4 mL) was added N-~2(R)-t-butoxycarbonylamino-3-triphenylmethyl mercapto-25 propyl]-L-proline (Fx~mple 1, Step C,~ (0.18 g, 0.34 mmol) followed by bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BOP-Cl) (0.175 g, 0.69 mmol) with stirring under argon at ambient temperature. After stirring for 20 h the solution was concentrated to dryness, and taken up în H2O
(30 mL) and extracted with EtOAc (3 x 20 mL). The organics were combined, washed with aq citric acid soln, aq satd NaHCO3 soln, brine 3 and dried (Na2SO4). Filtration and concentration followed by chromatography (sio2) (CH2C12: MeOH, 98:2)) provided the title compound. 1H NMR (CD30D) ~ 7.0-7.42 (m, 18H), 3.78-3.88 (m, lH), 3.06-3.19 (m, 2H), 2.14-2.7 (m, 6H), 2.26 (s, 3H), 1.93 (s, 3H), 1.66-1.99 (m, 3H), 1.41 (s, 9H).
WO 9G~'~,3~Z0 2 2 ~13 ~ ~ PCT/US95/12284 Step B: N-[2(R)-Amino-3-mercaptopropyl]-L-proline- 2,3-dimethylphenyl amide N-[2(R)-t-butoxycarbonylamino-3-triphenylmethyl-mercapto-propyl]-L-proline-2,3-dimethylphenyl amide (0.124 g, 0.19 mmol) was dissolved in CH2C12 (3 mL) and CF3C02H (1 mL) at ambient temperature, treated with triethylsilane (0.121 mL, 0.76 mmol) and stirred for 2 h. The reaction mixture was concentrated, triturated with 0.1 % aq TFA soln, the solid precipitate filtered off, and the filtrate lyophilized to provide the title compound. 1H NMR (CD30D) ~ 7.10 (s, 3H), 3.55-3.68 (m, lH), 3.32-3.45 (m, 2H), 2.75-3.19 (m, 5H), 2.5-2.65 (m, lH), 2.31 (s, 3H), 2.15 (s, 3H), 1.9-2.15 (m, 3H).
Anal. calcd for C16H25N30S-2.7 CF3CO2H: C, 41.77; H, 4.54; N, 6.83;
found: C, 41.70; H, 4.81; N, 7.17.
Using the methods described in Fx~mple 2 the following compounds were prepared:
N-[2(R)-Amino-3-mercaptopropyl]-L-3-trans-ethylproline-2,3-dimethylphenvl amide Anal. calcd for C1gH29N3OS-3 CF3CO2H: C, 42.54; H, 4.76; N, 6.20;foundC,42.16;H,5.06;N,6.64.
N-[2(R)-Amino-3-mercaptopropyl]-D-3-trans-ethylproline-2,3-dimethvlphenyl amide Anal. calcd for C1gH29N30S-2.5 CF3CO2H: C, 44.51; H, 5.12, N, 6.77; found C, 44.80; H, 5.19; N, 6.83.
In vitro inhibition of ras farnesvl transferase Assays offarnesyl-protein transferase. Partially purified bovine FPTase and Ras peptides (Ras-CVLS, Ras-CVIM and RAS-CAIL) 22~13 47 - 38 -were prepared as described by Schaber et al., J. Biol. Chem.
265:14701-14704 (1990), Pompliano, etal.,Biochemistry31:3800 (1992) and Gibbs et al., PNAS U.S~. 86:6630-6634 (1989). Bovine FPTase was assayed in a volume of 100 ,ul cont~ining 100 mM N-(2-hydroxy ethyl) piperazine-N'-(2-ethane sulfonic acid) (HEPES), pH
7.4, 5 mM MgCl2, 5 mM dithiothreitol (DTT), 100 mM [3H]-farnesyl diphosphate ([3H]-FPP; 740 CBq/mmol, New Fngl~ncl Nuclear), 650 nM Ras-CVLS and 10 ~g/ml FPTase at 31 C for 60 min. Reactions were initiated with FPTase and stopped with 1 ml of 1.0 M HCL in ethanol. Precipitates were collected onto filter-mats using a TomTec Mach II cell harvestor, washed with 100% ethanol, dried and counted in an LKB ,~-plate counter. The assay was linear with respect to both substrates, FPTase levels and time; less than 10% of the [3H]-F~PP
was utilized during the reaction period. Purified compounds were lS dissolved in 100% dimethyl sulfoxide (DMSO) and were diluted 20-fold into the assay. Percentage inhibition is measured by the amount of incorporation of radioactivity in the presence of the test compound when compared to the amount of incorporation in the absence of the test compound.
Human FPTase was prepared as described by Omer et al., Biochemistry 32:5167-5176 (1993). Human FPTase activity was assayed as described above with the exception that 0.1 % (w/v) polyethylene glycol 20,000, 10 ,llM ZnCl2 and 100 nM Ras-CVIM were added to the reaction mixture. Reactions were performed for 30 min., 2s stopped with 100 ,ul 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 inhibitory activity against hllm~n FPTase by the assay described above and were found to have IC50 of < 10 ,uM.
WO 9'1v3~20 PCT/US95112284 In vivo ras farnesvlation assay The cell line used in this assay is a v-ras line derived from 5 either Ratl or NIH3T3 cells, which expressed viral Ha-ras p21. The assay is performed essentially as described in DeClue, J.E. et al., Cancer Research 51:712-717, (1991). Cells in 10 cm dishes at 50-75%
confluency are treated with the test compound (final concentration of solvent, methanol or dimethyl sulfoxide, is 0.1 %). After 4 hours at 37C, the cells are labelled in 3 ml methionine-free DMEM supple-meted with 10% regular DMEM, 2% fetal bovine serum and 400 mCir35S]methionine (1000 Ci/mmol). After an additional 20 hours, the cells are lysed in 1 ml lysis buffer (1 % NP40/20 mM HEPES, pH 7.5/5 mM MgCl2/lmM DTT/10 mg/ml aprotinen/2 mg/ml leupeptin/2 mg/ml antipainlO.5 mM PMSF) and the lysates cleared by centrifugation at 100,000 x g for 45 min. Aliquots of lysates cont~inin~ equal nllmbers of acid-precipitable counts are bought to 1 ml with IP buffer (lysis buffer lacking DTT) and immunoprecipitated with the ras-specific monoclonal antibody Y13-259 (Furth, M.E. et al., J. Virol. 43:294-304, (1982)).
20 Following a 2 hour antibody incubation at 4C, 200 ml of a 25%
suspension of protein A-Sepharose coated with rabbit anti rat IgG is added for 45 min. The immlmoprecipitates are washed four times with IP
buffer (20 nM HEPES, pH 7.5/1 mM EDTA/1% Triton X-100Ø5%
deoxycholate/0.1 %/SDS/0.1 M NaCl) boiled in SDS-PAGE sample 25 buffer and loaded on 13% acrylamide gels. When the dye front reached the bottom, the gel is fixed, soaked in Fnligh~ening, dried and autoradiographed. The intensities of the bands corresponding to farnesylated and nonfarnesylated ras proteins are compared to determine the percent inhibition of farnesyl transfer to protein.
WO 9G~ X20 PCT/US95/12284 .
22013~7 EXAMPLE S
In vivo growth inhibition assav To determine the biological consequences of FPrase 5 inhibition, the effect of the compounds of the instant invention on the anchorage-independent growth of Ratl cells transformed with either a v-ras, v-raf, or v-mos oncogene is tested. Cells transformed by v Raf and v-Mos maybe included in the analysis to evaluate the specificity of instant compounds for Ras-induced cell transformation.
Rat 1 cells transformed with either v-ras, v-raf, or v-mos are seeded at a density of 1 x 104 cells per plate (35 mm in diameter) in a 0.3% top agarose layer in medium A (Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum) over a bottom agarose layer (0.6%). Both layers contain 0.1% methanol or an 15 a~ro~liate concentration of the instant compound (dissolved in methanol at 1000 times the final concentration used in the assay). The cells are fed twice weekly with O.S ml of medium A cont~ining 0.1% methanol or the concentration of the instant compound. Photomicrographs are taken 16 days after the cultures are seeded and comparisons are made.
N-r2(R)-Arnino-3-merca~to~lo~yll-L-prolirle-23-dimethylphenvl amide 20 Step A: N-r2(R)-t-butoxycarbonylamino-3-triphenyl methyl-mercaptopropvll-L-proline-2,3-dimethvlphenyl amide To a solution of 2,3-dimethylaniline (0.05 mL, 0.41 mmol) and diisopropylethylamine (0.12 mL, 0.69 mmol) in CH2Cl2 (4 mL) was added N-~2(R)-t-butoxycarbonylamino-3-triphenylmethyl mercapto-25 propyl]-L-proline (Fx~mple 1, Step C,~ (0.18 g, 0.34 mmol) followed by bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BOP-Cl) (0.175 g, 0.69 mmol) with stirring under argon at ambient temperature. After stirring for 20 h the solution was concentrated to dryness, and taken up în H2O
(30 mL) and extracted with EtOAc (3 x 20 mL). The organics were combined, washed with aq citric acid soln, aq satd NaHCO3 soln, brine 3 and dried (Na2SO4). Filtration and concentration followed by chromatography (sio2) (CH2C12: MeOH, 98:2)) provided the title compound. 1H NMR (CD30D) ~ 7.0-7.42 (m, 18H), 3.78-3.88 (m, lH), 3.06-3.19 (m, 2H), 2.14-2.7 (m, 6H), 2.26 (s, 3H), 1.93 (s, 3H), 1.66-1.99 (m, 3H), 1.41 (s, 9H).
WO 9G~'~,3~Z0 2 2 ~13 ~ ~ PCT/US95/12284 Step B: N-[2(R)-Amino-3-mercaptopropyl]-L-proline- 2,3-dimethylphenyl amide N-[2(R)-t-butoxycarbonylamino-3-triphenylmethyl-mercapto-propyl]-L-proline-2,3-dimethylphenyl amide (0.124 g, 0.19 mmol) was dissolved in CH2C12 (3 mL) and CF3C02H (1 mL) at ambient temperature, treated with triethylsilane (0.121 mL, 0.76 mmol) and stirred for 2 h. The reaction mixture was concentrated, triturated with 0.1 % aq TFA soln, the solid precipitate filtered off, and the filtrate lyophilized to provide the title compound. 1H NMR (CD30D) ~ 7.10 (s, 3H), 3.55-3.68 (m, lH), 3.32-3.45 (m, 2H), 2.75-3.19 (m, 5H), 2.5-2.65 (m, lH), 2.31 (s, 3H), 2.15 (s, 3H), 1.9-2.15 (m, 3H).
Anal. calcd for C16H25N30S-2.7 CF3CO2H: C, 41.77; H, 4.54; N, 6.83;
found: C, 41.70; H, 4.81; N, 7.17.
Using the methods described in Fx~mple 2 the following compounds were prepared:
N-[2(R)-Amino-3-mercaptopropyl]-L-3-trans-ethylproline-2,3-dimethylphenvl amide Anal. calcd for C1gH29N3OS-3 CF3CO2H: C, 42.54; H, 4.76; N, 6.20;foundC,42.16;H,5.06;N,6.64.
N-[2(R)-Amino-3-mercaptopropyl]-D-3-trans-ethylproline-2,3-dimethvlphenyl amide Anal. calcd for C1gH29N30S-2.5 CF3CO2H: C, 44.51; H, 5.12, N, 6.77; found C, 44.80; H, 5.19; N, 6.83.
In vitro inhibition of ras farnesvl transferase Assays offarnesyl-protein transferase. Partially purified bovine FPTase and Ras peptides (Ras-CVLS, Ras-CVIM and RAS-CAIL) 22~13 47 - 38 -were prepared as described by Schaber et al., J. Biol. Chem.
265:14701-14704 (1990), Pompliano, etal.,Biochemistry31:3800 (1992) and Gibbs et al., PNAS U.S~. 86:6630-6634 (1989). Bovine FPTase was assayed in a volume of 100 ,ul cont~ining 100 mM N-(2-hydroxy ethyl) piperazine-N'-(2-ethane sulfonic acid) (HEPES), pH
7.4, 5 mM MgCl2, 5 mM dithiothreitol (DTT), 100 mM [3H]-farnesyl diphosphate ([3H]-FPP; 740 CBq/mmol, New Fngl~ncl Nuclear), 650 nM Ras-CVLS and 10 ~g/ml FPTase at 31 C for 60 min. Reactions were initiated with FPTase and stopped with 1 ml of 1.0 M HCL in ethanol. Precipitates were collected onto filter-mats using a TomTec Mach II cell harvestor, washed with 100% ethanol, dried and counted in an LKB ,~-plate counter. The assay was linear with respect to both substrates, FPTase levels and time; less than 10% of the [3H]-F~PP
was utilized during the reaction period. Purified compounds were lS dissolved in 100% dimethyl sulfoxide (DMSO) and were diluted 20-fold into the assay. Percentage inhibition is measured by the amount of incorporation of radioactivity in the presence of the test compound when compared to the amount of incorporation in the absence of the test compound.
Human FPTase was prepared as described by Omer et al., Biochemistry 32:5167-5176 (1993). Human FPTase activity was assayed as described above with the exception that 0.1 % (w/v) polyethylene glycol 20,000, 10 ,llM ZnCl2 and 100 nM Ras-CVIM were added to the reaction mixture. Reactions were performed for 30 min., 2s stopped with 100 ,ul 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 inhibitory activity against hllm~n FPTase by the assay described above and were found to have IC50 of < 10 ,uM.
WO 9'1v3~20 PCT/US95112284 In vivo ras farnesvlation assay The cell line used in this assay is a v-ras line derived from 5 either Ratl or NIH3T3 cells, which expressed viral Ha-ras p21. The assay is performed essentially as described in DeClue, J.E. et al., Cancer Research 51:712-717, (1991). Cells in 10 cm dishes at 50-75%
confluency are treated with the test compound (final concentration of solvent, methanol or dimethyl sulfoxide, is 0.1 %). After 4 hours at 37C, the cells are labelled in 3 ml methionine-free DMEM supple-meted with 10% regular DMEM, 2% fetal bovine serum and 400 mCir35S]methionine (1000 Ci/mmol). After an additional 20 hours, the cells are lysed in 1 ml lysis buffer (1 % NP40/20 mM HEPES, pH 7.5/5 mM MgCl2/lmM DTT/10 mg/ml aprotinen/2 mg/ml leupeptin/2 mg/ml antipainlO.5 mM PMSF) and the lysates cleared by centrifugation at 100,000 x g for 45 min. Aliquots of lysates cont~inin~ equal nllmbers of acid-precipitable counts are bought to 1 ml with IP buffer (lysis buffer lacking DTT) and immunoprecipitated with the ras-specific monoclonal antibody Y13-259 (Furth, M.E. et al., J. Virol. 43:294-304, (1982)).
20 Following a 2 hour antibody incubation at 4C, 200 ml of a 25%
suspension of protein A-Sepharose coated with rabbit anti rat IgG is added for 45 min. The immlmoprecipitates are washed four times with IP
buffer (20 nM HEPES, pH 7.5/1 mM EDTA/1% Triton X-100Ø5%
deoxycholate/0.1 %/SDS/0.1 M NaCl) boiled in SDS-PAGE sample 25 buffer and loaded on 13% acrylamide gels. When the dye front reached the bottom, the gel is fixed, soaked in Fnligh~ening, dried and autoradiographed. The intensities of the bands corresponding to farnesylated and nonfarnesylated ras proteins are compared to determine the percent inhibition of farnesyl transfer to protein.
WO 9G~ X20 PCT/US95/12284 .
22013~7 EXAMPLE S
In vivo growth inhibition assav To determine the biological consequences of FPrase 5 inhibition, the effect of the compounds of the instant invention on the anchorage-independent growth of Ratl cells transformed with either a v-ras, v-raf, or v-mos oncogene is tested. Cells transformed by v Raf and v-Mos maybe included in the analysis to evaluate the specificity of instant compounds for Ras-induced cell transformation.
Rat 1 cells transformed with either v-ras, v-raf, or v-mos are seeded at a density of 1 x 104 cells per plate (35 mm in diameter) in a 0.3% top agarose layer in medium A (Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum) over a bottom agarose layer (0.6%). Both layers contain 0.1% methanol or an 15 a~ro~liate concentration of the instant compound (dissolved in methanol at 1000 times the final concentration used in the assay). The cells are fed twice weekly with O.S ml of medium A cont~ining 0.1% methanol or the concentration of the instant compound. Photomicrographs are taken 16 days after the cultures are seeded and comparisons are made.
Claims (16)
1. A compound which inhibits Ras farnesyl-transferase having the formula I:
wherein:
R1 is selected from:
a) hydrogen, b) R5S(O)2-, R5C(O)-, (R5)2NC(O)- or R6OC(O)-, and c) C1-C6 alkyl unsubstituted or substituted by aryl, heterocyclic, cycloalkyl, alkenyl, alkynyl, R5O-, R5S(O)m-, R5C(O)NR5-,CN,(R5)2N-C(NR5)-, R5C(O)-, R5OC(O)-,N3,-N(R5)2, or R6OC(O)NR5-;
R2a and R2b are independently selected from:
a) hydrogen, b) Cl-C6 alkyl unsubstituted or substituted by aryl, heterocycle, cycloalkyl, alkenyl, R5O-, R5S(O)m-, R5C(O)NR5-, CN,(R5)2N-C(NR5)-,R5C(O)-, R5OC(O)-,N3,-N(R5)2, or R6OC(O)NR5-, and c) aryl, heterocycle, cycloalkyl, alkenyl, R5O-, R5S(O)m-, R5C(O)NR5-,CN,N02,(R5)2N-C(NR5)-, R5C(O)-, R5OC(O)-,N3,-N(R5)2, or R6OC(O)NR5-, R3 is selected from:
a) unsubstituted or substituted aryl, b) unsubstituted or substituted heterocycle, c) unsubstituted or substituted cycloalkyl, and d) Cl-C6 alkyl substituted with hydrogen or an unsubstituted or substituted group selected from aryl, heterocycle and cycloalkyl;
X-Y is a) b) C) d) e) , or f) -CH2-CH2-;
R4a is selected from a) hydrogen, b) unsubstituted or substituted aryl, c) unsubstituted or substituted heterocycle, d) unsubstituted or substituted cycloalkyl, and e) Cl-C6 alkyl substituted with hydrogen or an unsubstituted or substituted group selected from aryl, heterocycle and cycloalkyl;
R4b is selected from a) hydrogen, b) unsubstituted or substituted aryl, c) unsubstituted or substituted heterocycle, d) unsubstituted or substituted cycloalkyl, e) C1-C6 alkyl substituted with hydrogen or an unsubstituted or substituted group selected from aryl, heterocycle and cycloalkyl, f) a carbonyl group which is bonded to an unsubstituted or substituted group selected from aryl, heterocycle, cycloalkyl and C1-C6 alkyl substituted with hydrogen or an unsubstituted or substituted group selected from aryl, heterocycle and cycloalkyl, and g) a sulfonyl group which is bonded to an unsubstituted or substituted group selected from aryl, heterocycle, cycloalkyl and C1-C6 alkyl substituted with hydrogen or an unsubstituted or substituted group selected from aryl, heterocycle and cycloalkyl;
R5 is independently selected from hydrogen, C1-C6 alkyl and aryl;
R6 is independently selected from C1-C6 alkyl and aryl;
Z is H2;
m is 0, 1 or 2, provided that m is 0 when R5 = hydrogen;
n is 0, 1,2,3 or 4; and t is.3, 4 or 5;
or a pharmaceutically acceptable salt thereof.
wherein:
R1 is selected from:
a) hydrogen, b) R5S(O)2-, R5C(O)-, (R5)2NC(O)- or R6OC(O)-, and c) C1-C6 alkyl unsubstituted or substituted by aryl, heterocyclic, cycloalkyl, alkenyl, alkynyl, R5O-, R5S(O)m-, R5C(O)NR5-,CN,(R5)2N-C(NR5)-, R5C(O)-, R5OC(O)-,N3,-N(R5)2, or R6OC(O)NR5-;
R2a and R2b are independently selected from:
a) hydrogen, b) Cl-C6 alkyl unsubstituted or substituted by aryl, heterocycle, cycloalkyl, alkenyl, R5O-, R5S(O)m-, R5C(O)NR5-, CN,(R5)2N-C(NR5)-,R5C(O)-, R5OC(O)-,N3,-N(R5)2, or R6OC(O)NR5-, and c) aryl, heterocycle, cycloalkyl, alkenyl, R5O-, R5S(O)m-, R5C(O)NR5-,CN,N02,(R5)2N-C(NR5)-, R5C(O)-, R5OC(O)-,N3,-N(R5)2, or R6OC(O)NR5-, R3 is selected from:
a) unsubstituted or substituted aryl, b) unsubstituted or substituted heterocycle, c) unsubstituted or substituted cycloalkyl, and d) Cl-C6 alkyl substituted with hydrogen or an unsubstituted or substituted group selected from aryl, heterocycle and cycloalkyl;
X-Y is a) b) C) d) e) , or f) -CH2-CH2-;
R4a is selected from a) hydrogen, b) unsubstituted or substituted aryl, c) unsubstituted or substituted heterocycle, d) unsubstituted or substituted cycloalkyl, and e) Cl-C6 alkyl substituted with hydrogen or an unsubstituted or substituted group selected from aryl, heterocycle and cycloalkyl;
R4b is selected from a) hydrogen, b) unsubstituted or substituted aryl, c) unsubstituted or substituted heterocycle, d) unsubstituted or substituted cycloalkyl, e) C1-C6 alkyl substituted with hydrogen or an unsubstituted or substituted group selected from aryl, heterocycle and cycloalkyl, f) a carbonyl group which is bonded to an unsubstituted or substituted group selected from aryl, heterocycle, cycloalkyl and C1-C6 alkyl substituted with hydrogen or an unsubstituted or substituted group selected from aryl, heterocycle and cycloalkyl, and g) a sulfonyl group which is bonded to an unsubstituted or substituted group selected from aryl, heterocycle, cycloalkyl and C1-C6 alkyl substituted with hydrogen or an unsubstituted or substituted group selected from aryl, heterocycle and cycloalkyl;
R5 is independently selected from hydrogen, C1-C6 alkyl and aryl;
R6 is independently selected from C1-C6 alkyl and aryl;
Z is H2;
m is 0, 1 or 2, provided that m is 0 when R5 = hydrogen;
n is 0, 1,2,3 or 4; and t is.3, 4 or 5;
or a pharmaceutically acceptable salt thereof.
2. The compound according to Claim 1 having a structure of the formula I:
I
wherein:
R1 is selected from:
a) hydrogen, b) R5S(O)2-, R5C(O)-, (R5)2NC(O)- or R6OC(O)-, and c) C1-C6 alkyl unsubstituted or substituted by aryl, heterocyclic, cycloalkyl, alkenyl, alkynyl, R5O-, R5S(O)m-, R5C(O)NR5-, CN,(R5)2N-C(NR5)-, R5C(O)-, R5OC(O)-,N3,-N(R5)2,or R6C(O)NR5-;
Ra and Rb are independently selected from:
a) hydrogen, b) Cl-C6 alkyl unsubstitudes or substitudes by aryl, heterocycle, cycloalkyl, alkenyl, R5O-, R5S(O)m-, R5C(O)NR5-, CN, (R5)2N-C(NR5)-, R5C(O)-, R5OC(O)-, N3, -N(R5)2, or R6OC(O)NR5-, and c) aryl, heterocycle, cycloalkyl, alkenyl, R5O-, R5S(O)m-, R5C(O)NR5-, CN, NO2. (R5)2N-C(NR5)-, R5C(O)-, R5OC(O)-, N3,-N(R5)2, or R6OC(O)NR5-, R3 is selected from:
a) unsubstituted or substituted aryl, b) unsubstituted or substituted heterocycle, c) insubstituted or substituted cycloalkyl, and d) Cl-C6 alkyl substituted with an unsubstituted or substituted group selected from aryl, heterocycle and cycloalkyl;
X-Y is a) , or b) R4a is selected from a) hydrogen, b) unsubstituted or substituted aryl, c) unsubstituted or substituted heterocycle, d) unsubstituted or substituted cycloalkyl, and e) Cl-C6 alkyl substituted with hydrogen or an unsubstituted or substituted group selected from aryl, heterocycle and cycloalkyl;
wherein heterocycle is selected from pyrrolidinyl, imidazolyl, pyridinyl, thiazolyl, pyridonyl, 2-oxopiperidinyl, indolyl, quinolinyl, isoquinolinyl, and thienyl;
R4b is selected from a) hydrogen, b) unsubstituted or substituted aryl, c) unsubstituted or substituted heterocycle, d) unsubstituted or substituted cycloalkyl, e) Cl-C6 alkyl substituted with hydrogen or an unsubstituted or substituted group selected from aryl, heterocycle and cycloalkyl, f) a carbonyl group which is bonded to an unsubstituted or substituted group selected from aryl, heterocycle, cycloalkyl and Cl-C6 alkyl substituted with hydrogen or an unsubstituted or substituted group selected from aryl, heterocycle and cycloalkyl, and g) a sulfonyl group which is bonded to an unsubstituted or substituted group selected from aryl, heterocycle, cycloalkyl and C1-C6 alkyl substituted with hydrogen or an unsubstituted or substituted group selected from aryl, heterocycle and cycloalkyl;
wherein heterocycle is selected from pyrrolidinyl, imidazolyl, pyridinyl, thiazolyl, pyridonyl, 2-oxopiperidinyl, indolyl, quinolinyl, isoquinolinyl, and thienyl;
R5 is independently selected from hydrogen, C1-C6 alkyl and aryl;
R6 is independently selected from C1-C6 alkyl and aryl;
Z is H2;
m is 0, 1 or 2, provided that m is 0 when R5 = hydrogen;
n is 0, 1, 2, 3 or 4; and t is 3, 4 or 5;
or a pharmaceutically acceptable salt thereof.
I
wherein:
R1 is selected from:
a) hydrogen, b) R5S(O)2-, R5C(O)-, (R5)2NC(O)- or R6OC(O)-, and c) C1-C6 alkyl unsubstituted or substituted by aryl, heterocyclic, cycloalkyl, alkenyl, alkynyl, R5O-, R5S(O)m-, R5C(O)NR5-, CN,(R5)2N-C(NR5)-, R5C(O)-, R5OC(O)-,N3,-N(R5)2,or R6C(O)NR5-;
Ra and Rb are independently selected from:
a) hydrogen, b) Cl-C6 alkyl unsubstitudes or substitudes by aryl, heterocycle, cycloalkyl, alkenyl, R5O-, R5S(O)m-, R5C(O)NR5-, CN, (R5)2N-C(NR5)-, R5C(O)-, R5OC(O)-, N3, -N(R5)2, or R6OC(O)NR5-, and c) aryl, heterocycle, cycloalkyl, alkenyl, R5O-, R5S(O)m-, R5C(O)NR5-, CN, NO2. (R5)2N-C(NR5)-, R5C(O)-, R5OC(O)-, N3,-N(R5)2, or R6OC(O)NR5-, R3 is selected from:
a) unsubstituted or substituted aryl, b) unsubstituted or substituted heterocycle, c) insubstituted or substituted cycloalkyl, and d) Cl-C6 alkyl substituted with an unsubstituted or substituted group selected from aryl, heterocycle and cycloalkyl;
X-Y is a) , or b) R4a is selected from a) hydrogen, b) unsubstituted or substituted aryl, c) unsubstituted or substituted heterocycle, d) unsubstituted or substituted cycloalkyl, and e) Cl-C6 alkyl substituted with hydrogen or an unsubstituted or substituted group selected from aryl, heterocycle and cycloalkyl;
wherein heterocycle is selected from pyrrolidinyl, imidazolyl, pyridinyl, thiazolyl, pyridonyl, 2-oxopiperidinyl, indolyl, quinolinyl, isoquinolinyl, and thienyl;
R4b is selected from a) hydrogen, b) unsubstituted or substituted aryl, c) unsubstituted or substituted heterocycle, d) unsubstituted or substituted cycloalkyl, e) Cl-C6 alkyl substituted with hydrogen or an unsubstituted or substituted group selected from aryl, heterocycle and cycloalkyl, f) a carbonyl group which is bonded to an unsubstituted or substituted group selected from aryl, heterocycle, cycloalkyl and Cl-C6 alkyl substituted with hydrogen or an unsubstituted or substituted group selected from aryl, heterocycle and cycloalkyl, and g) a sulfonyl group which is bonded to an unsubstituted or substituted group selected from aryl, heterocycle, cycloalkyl and C1-C6 alkyl substituted with hydrogen or an unsubstituted or substituted group selected from aryl, heterocycle and cycloalkyl;
wherein heterocycle is selected from pyrrolidinyl, imidazolyl, pyridinyl, thiazolyl, pyridonyl, 2-oxopiperidinyl, indolyl, quinolinyl, isoquinolinyl, and thienyl;
R5 is independently selected from hydrogen, C1-C6 alkyl and aryl;
R6 is independently selected from C1-C6 alkyl and aryl;
Z is H2;
m is 0, 1 or 2, provided that m is 0 when R5 = hydrogen;
n is 0, 1, 2, 3 or 4; and t is 3, 4 or 5;
or a pharmaceutically acceptable salt thereof.
3. A compound which inhibits farnesyl-protein transferase which is:
N-[2(R)-Amino-3-mercaptopropyl]-L-proline-2,3-dichlorobenzylamide N-[2(R)-Amino-3-mercaptopropyl]-L-proline-1-naphthylmethyl amide N-[2(R)-Amino-3-mercaptopropyl]-L-pipecolyl-2,3-dichlorobenzamide N-[2(R)-Amino-3 -mercaptopropyl]-L-3-trans-ethylproline-2, 3-dichlorobenzamide N-[2(R)-Amino-3-mercaptopropyl]-D-3-trans-ethylproline-2, 3-dichloro-benzamide N-[2(R)-Amino-3-mercaptopropyl]-L-3-cis-ethylproline-2, 3-dichloro-benzamide N-[2(R)-Amino-3-mercaptopropyl]-D-3-cis-ethylproline-2, 3-dichloro-benzamide N-[2(R)-Amino-3 -mercaptopropyl]-L-3-trans-ethylproline-1-naphthyl-methyl amide N-[2(R)-Amino-3-mercaptopropyl]-D-3-trans-ethylproline-1-naphthyl-methyl amide N-[2(R)-Amino-3-mercaptopropyl]-L-proline-2,3-dimethylphenyl amide N-[2(R)-Amino-3-mercaptopropyl]-L-3-trans-ethylproline-2, 3-dimethylphenyl amide, or N-[2(R)-Amino-3-mercaptopropyl]-D-3-trans-ethylproline-2, 3-dimethylphenyl amide or a pharmaceutically acceptable salt thereof.
N-[2(R)-Amino-3-mercaptopropyl]-L-proline-2,3-dichlorobenzylamide N-[2(R)-Amino-3-mercaptopropyl]-L-proline-1-naphthylmethyl amide N-[2(R)-Amino-3-mercaptopropyl]-L-pipecolyl-2,3-dichlorobenzamide N-[2(R)-Amino-3 -mercaptopropyl]-L-3-trans-ethylproline-2, 3-dichlorobenzamide N-[2(R)-Amino-3-mercaptopropyl]-D-3-trans-ethylproline-2, 3-dichloro-benzamide N-[2(R)-Amino-3-mercaptopropyl]-L-3-cis-ethylproline-2, 3-dichloro-benzamide N-[2(R)-Amino-3-mercaptopropyl]-D-3-cis-ethylproline-2, 3-dichloro-benzamide N-[2(R)-Amino-3 -mercaptopropyl]-L-3-trans-ethylproline-1-naphthyl-methyl amide N-[2(R)-Amino-3-mercaptopropyl]-D-3-trans-ethylproline-1-naphthyl-methyl amide N-[2(R)-Amino-3-mercaptopropyl]-L-proline-2,3-dimethylphenyl amide N-[2(R)-Amino-3-mercaptopropyl]-L-3-trans-ethylproline-2, 3-dimethylphenyl amide, or N-[2(R)-Amino-3-mercaptopropyl]-D-3-trans-ethylproline-2, 3-dimethylphenyl amide or a pharmaceutically acceptable salt thereof.
4. The compound according to Claim 3 which inhibits farnesyl-protein transferase which is:
N-[2(R)-Amino-3-mercaptopropyl]-L-3-trans-ethylproline-2, 3-dichlorobenzamide or a pharmaceutically acceptable salt thereof.
N-[2(R)-Amino-3-mercaptopropyl]-L-3-trans-ethylproline-2, 3-dichlorobenzamide or a pharmaceutically acceptable salt thereof.
5. The compound according to Claim 3 which inhibits farnesyl-protein transferase which is:
N-[2(R)-Amino-3-mercaptopropyl]-L-3-cis-ethylproline-2, 3-dichlorobenzamide or a pharmaceutically acceptable salt thereof.
N-[2(R)-Amino-3-mercaptopropyl]-L-3-cis-ethylproline-2, 3-dichlorobenzamide or a pharmaceutically acceptable salt thereof.
6. The compound according to Claim 3 which inhibits farnesyl-protein transferase which is:
N-[2(R)-Amino-3-mercaptopropyl]-L-3-trans-ethylproline-1-naphthylmethyl amide or a pharmaceutically acceptable salt thereof.
N-[2(R)-Amino-3-mercaptopropyl]-L-3-trans-ethylproline-1-naphthylmethyl amide or a pharmaceutically acceptable salt thereof.
7. The compound according to Claim 3 which inhibits farnesyl-protein transferase which is:
N-[2(R)-Amino-3-mercaptopropyl]-L-3-trans-ethylproline-2, 3-dimethylphenyl amide or a pharmaceutically acceptable salt thereof.
N-[2(R)-Amino-3-mercaptopropyl]-L-3-trans-ethylproline-2, 3-dimethylphenyl amide or a pharmaceutically acceptable salt thereof.
8. A pharmaceutical composition comprising a pharmaceutical carrier, and dispersed therein, a therapeutically effective amount of a compound of Claim 1.
9. A pharmaceutical composition comprising a pharmaceutical carrier, and dispersed therein, a therapeutically effective amount of a compound of Claim 2.
10. A pharmaceutical composition comprising a pharmaceutical carrier, and dispersed therein, a therapeutically effective amount of a compound of Claim 3.
11. A method for inhibiting farnesylation of Ras protein which comprises administering to a mammal in need thereof a therapeutically effective amount of the composition of Claim 8.
12. A method for inhibiting farnesylation of Ras protein which comprises administering to a mammal in need thereof a therapeutically effective amount of the composition of Claim 9.
13. A method for inhibiting farnesylation of Ras protein which comprises administering to a mammal in need thereof a therapeutically effective amount of the composition of Claim 10.
14. A method for treating cancer which comprises administering to a mammal in need thereof a therapeutically effective amount of a composition of Claim 8.
15. A method for treating cancer which comprises administering to a mammal in need thereof a therapeutically effective amount of a composition of Claim 9.
16. A method for treating cancer which comprises administering to a mammal in need thereof a therapeutically effective amount of a composition of Claim 10.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US08/315,151 US5491164A (en) | 1994-09-29 | 1994-09-29 | Inhibitors of farnesyl-protein transferase |
| US315,151 | 1994-09-29 | ||
| PCT/US1995/012284 WO1996009820A1 (en) | 1994-09-29 | 1995-09-25 | Inhibitors of farnesyl-protein transferase |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2201347A1 true CA2201347A1 (en) | 1996-04-04 |
Family
ID=29405753
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2201347 Abandoned CA2201347A1 (en) | 1994-09-29 | 1995-09-25 | Inhibitors of farnesyl-protein transferase |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA2201347A1 (en) |
-
1995
- 1995-09-25 CA CA 2201347 patent/CA2201347A1/en not_active Abandoned
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