Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
  • C07D233/54—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
  • C07D233/64—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with substituted hydrocarbon radicals attached to ring carbon atoms, e.g. histidine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
  • C07C323/50—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton
  • C07C323/51—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton
  • C07C323/57—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being further substituted by nitrogen atoms, not being part of nitro or nitroso groups
  • C07C323/58—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being further substituted by nitrogen atoms, not being part of nitro or nitroso groups with amino groups bound to the carbon skeleton
  • C07C323/59—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being further substituted by nitrogen atoms, not being part of nitro or nitroso groups with amino groups bound to the carbon skeleton with acylated amino groups bound to the carbon skeleton

Definitions

• Ras protein is part of a signalling pathway that links cell surface growth factor receptors to nuclear signals initiating cellular proliferation.
• Biological and biochemical studies of Ras action indicate that Ras functions like a G-regulatory protein.
• Ras In the inactive state, Ras is bound to GDP.
• Ras Upon growth factor receptor activation Ras is induced to exchange GDP for GTP and undergoes a conformational change.
• the GTP-bound form of Ras propagates the growth stimulatory signal until the signed is terminated by the intrinsic GTPase activity of Ras, which returns the protein to its inactive GDP bound form (D.R. Lowy and D.M. Willumsen, Ann. Rev. Biochem. 62:851-891 (1993)).
• Mutated ras genes are found in many human cancers, including colorectal carcinoma, exocrine pancreatic carcinoma, and myeloid leukemias.
• the protein products of these genes are defective in their GTPase activity and constitutively transmit a growth stimulatory signal.
• Ras must be localized to the plasma membrane for both normal and oncogenic functions. At least 3 post-translational modifications are involved with Ras membrane localization, and all 3 modifications occur at the C-terminus of Ras.
• the Ras C-terminus contains a sequence motif termed a "CAAX” or "Cys-Aaa ⁇ -Aaa ⁇ -Xaa” box (Cys is cysteine, Aaa is an aliphatic amino acid, the Xaa is any amino acid) (Willumsen et al., Nature 370:583-586 (1984)).
• this motif serves as a signal sequence for the enzymes famesyl-protein transferase or geranylgeranyl-protein transferase, which catalyze the alkylation of the cysteine residue of the CAAX motif with a C15 or C20 isoprenoid, respectively.
• the Ras protein is one of several proteins that are known to undergo post-translational famesylation. Other farnesylated proteins.include the Ras-related GTP-binding proteins such as Rho, fungal mating factors, the nuclear lamins, and the gamma subunit of transducin. James, et al., J. Biol. Chem. 269, 14182 (1994) have identified a peroxisome associated protein Pxf which is also farnesylated.
• HMG-CoA reductase the rate limiting enzyme for the production of polyisoprenoids
• famesyl pyrophosphate utilizes famesyl pyrophosphate to covalently modify the Cys thiol group of the Ras CAAX box with a famesyl group (Reiss et al, Cell, 62:81-88 (1990); Schaber et al, J. Biol Chem., 265:14701-14704 (1990); Schafer et al, Science, 249:1133-1139 (1990); Manne et al, Proc. Natl Acad. Sci USA, £7:7541-7545 (1990)).
• Inhibition of famesyl pyrophosphate biosynthesis by inhibiting HMG-CoA reductase blocks Ras membrane localization in cultured cells.
• direct inhibition of famesyl-protein transferase would be more specific and attended by fewer side effects than would occur with the required dose of a general inhibitor of isoprene biosynthesis.
• FPTase famesyl-protein transferase
• FPP famesyl diphosphate
• Ras protein substrates
• peptide derived class of inhibitors a subclass of inhibitors has been described which generally comprises cysteine containing molecules that are related to the CAAX motif that is the signal for protein prenylation.
• Such inhibitors may inhibit protein prenylation while serving as alternate substrates for the famesyl-protein transferase enzyme, or may be purely competitive inhibitors (U.S. Patent 5,141,851, University of Texas; N.E. Kohl et al, Science, 260:1934-1937 (1993); Graham, et al., J. Med. Chem., 37, 725 (1994)).
• deletion of the thiol from a peptidyl CAAX derivative has been shown to dramatically reduce the inhibitory potency of the compound.
• the thiol group potentially places limitations on the therapeutic application of FPTase inhibitors with respect to pharmacokinetics, pharmacodynamics and toxicity. Therefore, a functional replacement for the thiol is desirable.
• peptide derived inhibitors which comprises peptidomimetic compounds wherein the central AA portion of the CAAX motif has been replaced by 3-aminobenzoic acid and 3- aminomethylbenzoic acid spacers has recently been described (M. Nigam et al. J. Biol. Chem., 265:20695-20698 (1993), Y. Qian et al. /. Biol. Chem., 269: 12410-12413 (1994)).
• FPTase peptidomimetic inhibitors further lacking a C-terminus peptidyl moiety wherein the X peptide has been replaced by a non-peptide moiety have also been recendy described (A. Vogt et al. J. BiolChem., 270:660-664 (1995)). All of the compounds in this second subclass of peptide derived inhibitors retain the thiol moiety.
• the present invention includes substituted aminoalkylbenza ⁇ iide and aminobenzamide analogs which inhibit the famesyl-protein transferase, chemotherapeutic compositions containing the compounds of this invention, and methods for producing the compounds of this invention. Furthermore these analogs differ from those previously described as inhibitors of famesyl-protein transferase in that they do not have a thiol moiety. The lack of the thiol offers unique advantages in terms of improved pharmacokinetic behavior in animals, prevention of thiol-dependent chemical reactions, such as rapid autoxidation and disulfide formation with endogenous thiols, and reduced systemic toxicity.
• the compounds of this invention are useful in the inhibition of famesyl-protein transferase and the famesylation of certain proteins.
• the famesyl-protein transferase inhibitors are illustrated by the formula I:
• Rla and Rib are independently selected from: a) hydrogen, b) aryl, heterocycle, cycloalkyl, alkenyl, alkynyl, RI ⁇ O-, RHS(0)m-, Rl°C(O)NRl0-, CN, N ⁇ 2, (RlO) 2 N-C(NRl )-, RlOC(O)-, Rl ⁇ C(O)-, N3, -N(Rl )2, or RH ⁇ C(O)NRl0-, c) C1-C6 alkyl unsubstituted or substituted by aryl, heterocyclic, cycloalkyl, alkenyl, alkynyl, R ⁇ O-, R ⁇ S(0) m -, Rl°C(O)NRl0-, CN, (R1 )2N-C(NR1 )-, RlOC(O)-, RlO ⁇ C(O)-, N3, -N(Rl ) 2 , or
• R2a and R2b are independently selected from: a) hydrogen, b) C 1-C6 alkyl unsubstituted or substituted by alkenyl, R IOO-,
• R3a and R3b are independently selected from: a) a side chain of a naturally occurring amino acid, b) an oxidized form of a side chain of a naturally occurring amino acid which is: i) methionine sulfoxide, or ii) methionine sulfone, and c) substituted or unsubstituted C1-C2O alkyl, C2-C2O alkenyl, C3-C10 cycloalkyl, aryl or heterocyclic group, wherein the substituent is selected from F, Cl, Br, N(RlO)2, N02, R 10 O-, Rl lS(0) m -, R1°C(0)NR1 -, CN, (R
• R3a and R3b are combined to form - (CH2)s - wherein one of the carbon atoms is optionally replaced by a moiety selected from: O, S(0)m, -NC(O)-, and -N(CORl )- ;
• R4 and R5 are independently selected from: a) hydrogen, and
• R7 is independently selected from: a) hydrogen, b) aryl, heterocycle, cycloalkyl, alkenyl, alkynyl, perfluoroalkyl, F, Cl, Br, RlOO-, RHS(0) m -, Rl c(O)NRl0-, CN, N02, Rl ⁇ 2N-C(NRlO)-, RlOC(O)-, RlO ⁇ C(O)-, N3, -N(RlO)2, or RH ⁇ C(O)NRl -, and c) C1-C6 alkyl unsubstituted or substituted by aryl, heterocycle, cycloalkyl, alkenyl, alkynyl, perfluoroalkyl, F, Cl, Br, RlOO-, RllS(0) m -, Rl°C(0)NH-, CN, H2N- C(NH)-, Rl C(O)-, RlO ⁇ C(O)-,
• R8 is selected from: a) hydrogen, b) alkenyl, alkynyl, perfluoroalkyl, F, Cl, Br, Rl O-, RllS(0) m -, Rl°C(O)NRl0-, CN, N02,
• RlO is independently selected from hydrogen, C1-C6 alkyl, benzyl and aryl;
• Rl 1 is independently selected from C1-C6 alkyl and aryl
• V is not hydrogen if Al is S(0)m and V is not hydrogen if A is a bond, n is 0 and A ⁇ is S(0) ;
• W is a heterocycle
• Z is independently H2 or O
• n 0, 1, 2, 3 or 4
• p 0, 1, 2, 3 or 4
• r 0 to 5, provided that r is 0 when V is hydrogen; s is 4 or 5; and u is O or l; or the pharmaceutically acceptable salts thereof.
• Rla and Rib are independently selected from: a) hydrogen, b) aryl, heterocycle, cycloalkyl, alkenyl, alkynyl, R OO-, Rl lS(0) ⁇ r, R 10 C(O)NRl0_.
• CN N02, (Rl )2N-C(NRlO)-, RlOC(O)-, RlO ⁇ C(O)-, N3, -N(RlO)2, or RH ⁇ C(O)NRl0-, c) C l -C6 alkyl unsubstituted or substituted by aryl, heterocyclic, cycloalkyl, alkenyl, alkynyl, Rl ⁇ O-, R n S(0)m-, Rl°C(O)NRl0-, CN, (R1 ) 2 N-C(NR10)-, RlOC(O)-, Rl ⁇ C(O)-, N3, -N(RlO)2, or Rl l ⁇ C(0> NR10-;
• R2 and R2b are independently selected from: a) hydrogen, b) C l -C6 alkyl unsubstituted or substituted by alkenyl, R 0 ⁇ -, R n S(0) ⁇ r, Rl°C(O)NRl0-, CN, N3, (R10)2N-C(NR10)-, RlOC(O)-, RlO ⁇ C(O)-, -N(RlO)2, or RHOC(O)NR1 -, c) aryl, heterocycle, cycloalkyl, alkenyl, Rl ⁇ O-,
• R3a and R3b are independently selected from: a) a side chain of a naturally occurring amino acid, b) an oxidized form of a side chain of a naturally occurring amino acid which is: i) methionine sulfoxide, or ii) methionine sulfone, and c) substituted or unsubstituted C 1-C20 alkyl, C2-C20 alkenyl,
• R4 and R5 are independently selected from: a) hydrogen, and
• R6 is a) substituted or unsubstituted Cl-C8 alkyl or substituted or unsubstituted C5-C8 cycloalkyl, wherein the substituent on the alkyl is selected from:
• R7 is independently selected from: a) hydrogen, b) aryl, heterocycle, cycloalkyl, alkenyl, alkynyl, perfluoroalkyl, F, Cl, Br, RlOO-, Rl lS(0) m -, Rl C(O)NRl -, CN, N02, Rl ⁇ 2N-C(NRlO)-, Rl°C(0)-,
• R8 is selected from: a) hydrogen, b) alkenyl, alkynyl, perfluoroalkyl, F, Cl, Br, RlOO-, RllS(0) m -, Rl°C(O)NRl -, CN, NO2, (RlO)2N-C-(NRl )-, RlOc(O)-, Rl ⁇ C(O)-, N3, -N(RlO)2, or RHOC(O)NR1 -, and c) C 1 -C6 alkyl unsubstituted or substituted by perfluoroalkyl,
• RlO is independently selected from hydrogen, C1-C6 alkyl, benzyl and aryl;
• Rl 1 is independently selected from C1-C6 alkyl and aryl
• Rl is independently selected from hydrogen and -C alkyl
• Rl3 is independently selected from C1-C6 alkyl
• V is not hydrogen if A 1 is S(0)m and V is not hydrogen if Al is a bond, n is 0 and A2 is S(0)m;
• W is a heterocycle
• Z is independently H2 or O
• Rla and Rib are independently selected from: a) hydrogen, b) aryl, heterocycle, cycloalkyl, alkenyl, alkynyl, RlOO-, R 1 !S(0) m -, Rl°C(O)NRl0-, CN, N ⁇ 2, (Rl ) 2 N-C(NRlO)-, RlOC(O)-, RlO ⁇ C(O)-, N3, -N(Rl ) 2 , or Rl lOC(O)NRl0-, c) C1-C6 alkyl unsubstituted or substituted by aryl, heterocyclic, cycloalkyl, alkenyl, alkynyl, RI ⁇ O-, Rl lS(0) m -, Rl0C(O)NRl -, CN, (RlO)2N-C(NRlO)-, RlOC(O)-, RlO ⁇ C(O)-, N3, -N
• R2a and R2b are independently selected from: a) hydrogen, b) C 1 -C6 alkyl unsubstituted or substituted by alkenyl, R 1 OO- , Rl lS(0)m-, Rl°C(O)NRl0-, CN, N3, (RlO)2N-C(NRlO)-,
• R4 and R5 are independently selected from: a) hydrogen, and
• R7 is independently selected from: a) hydrogen, b) aryl, heterocycle, cycloalkyl, alkenyl, alkynyl, perfluoroalkyl, F, Cl, Br, RlOO-, Rl lS(0) m -, RlOC(0)NRlO-, CN, N02, Rl°2N-C(NRl )-, R10C(O)-, RlO ⁇ C(O)-, N3, -N(RlO) 2 , or RHOC(0)NR1 -, and c) C l -C6 alkyl unsubstituted or substituted by aryl, heterocycle, cycloalkyl, alkenyl, alkynyl, perfluoroalkyl, F, Cl, Br, RlOO-, RHS(0) m -, Rl0C(O)NH-, CN, H2N-C(NH)-, Rl C(O)-, RlO ⁇ C(O
• R8 is selected from: a) hydrogen, b) alkenyl, alkynyl, perfluoroalkyl, F, Cl, Br, R IOO-, RllS(0) ⁇ r, R1°C(0)NR10-, CN, N02, (R i0 )2N-C-
• RlO is independently selected from hydrogen, C1-C6 alkyl, benzyl and aryl;
• RU is independently selected from C1-C6 alkyl and aryl
• V is not hydrogen if A 1 is S(0)m and V is not hydrogen if Al is a bond, n is 0 and A2 is S(0) m ;
• Rla and Ri are independently selected from: a) hydrogen, b) aryl, heterocycle, cycloalkyl, alkenyl, alkynyl, Rl ⁇ O-,
• R2a and R2b are independendy selected from: a) hydrogen, b) Cl-C6 alkyl unsubstituted or substituted by alkenyl, Rl ⁇ O-, Rl lS(0)m-, Rl°C(O)NRl0-, CN, N3, (R10) 2 N-C(NR10)-,
• R4 and R5 are independendy selected from: a) hydrogen, and
• R7 is independently selected from: a) hydrogen, b) aryl, heterocycle, cycloalkyl, alkenyl, alkynyl, perfluoroalkyl, F, Cl, Br, RlOO-, Rl !S(0) m -, R10C(O)NR10-, CN, N ⁇ 2, Rl ⁇ 2N-C(NRlO)-, RlOC(O)-, RlO ⁇ C(O)-, N3, -N(RlO)2, or Rl l ⁇ C(O)NRl0-, and c) C1-C6 alkyl unsubstituted or substituted by aryl, heterocycle, cycloalkyl, alkenyl, alkynyl, perfluoroalkyl, F, Cl, Br, RlOO-, RHS(0)m-, Rl°C(0)NH-, CN, H2N- C(NH)-, RlOC(O)-, RlO ⁇ C(O)-,
• R8 is selected from: a) hydrogen, b) alkenyl, alkynyl, perfluoroalkyl, F, Cl, Br, R IOO-, Rl lS(0)m-, Rl°C(O)NRl0-, CN, N ⁇ 2, (RlO)2N-C-(NRlO)-, RlOc(O)-, RIOOC(O)-, N3, -N(RlO)2, or Rl 10C(0)NR10-, and c) C 1 -C6 alkyl unsubstituted or substituted by perfluoroalkyl, F, Cl, Br, RlOO-, RHS(0) m -, R1°C(0)NR10-, CN, (RlO) 2 N-C(NRl )-, RlOc(O)-, Rl ⁇ C(O)-, N3, -N(RlO) 2 , O ⁇ RHOC(O)NR10-;
• RlO is independendy selected from hydrogen, C1-C6 alkyl, benzyl and aryl;
• Rl 1 is independendy selected from C1-C6 alkyl and aryl
• V is selected from: a) hydrogen, b) heterocycle, c) aryl, d) C1-C2O alkyl wherein from 0 to 4 carbon atoms are replaced with a a heteroatom selected from O, S, and N, and e) C2-C2O alkenyl, provided that V is not hydrogen if Al is S(0)m and V is not hydrogen if Al is a bond, n is 0 and A2 is S(0)m; W is a heterocycle;
• Z is independently H2 or O
• Ras famesyl transferase inhibitors are illustrated by the Formula la:
• Rla is independendy selected from: hydrogen or Cl-C6 alkyl
• Rib is independendy selected from: a) hydrogen, b) aryl, heterocycle, cycloalkyl, RlOO-, -N(RlO)2 or alkenyl, c) C 1-C6 alkyl unsubstituted or substituted by aryl, heterocycle, cycloalkyl, alkenyl, RlOO-, or -N(RlO)2;
• R2a is selected from: a) hydrogen, b) C 1 -C6 alkyl unsubstituted or substituted by alkenyl, R 10 ⁇ -, Rl lS(0)m- > Rl°C(O)NRl0-, CN, N3, (R1 ) 2 N-C(NR1 )-, Rl C(O)-, RlO ⁇ C(O)-, -N(Rl )2, or RHOC(O)NR10-, c) aryl, heterocycle, cycloalkyl, alkenyl, Rl ⁇ O-, Rl lS(0) m -, R10C(O)NR10-, CN, N ⁇ 2,
• R2b is hydrogen
• R3a and R3b are independendy selected from: a) a side chain of a naturally occurring amino acid, b) an oxidized form of a side chain of a naturally occurring amino acid which is: i) methionine sulfoxide, or ii) methionine sulfone, c) substituted or unsubstituted C 1 -C 10 alkyl, C2-C 10 alkenyl,
• C3-C10 cycloalkyl, aryl or heterocyclic group wherein the substituent is selected from F, Cl, Br, N ⁇ 2, Rl°0-, Rl lS(0)m-, R1°C(0)NR10-, CN, (RlO) 2 N-C(NRl )-, RlOC(O)-, Rl ⁇ C(O)-, N3, -N(RlO)2, Rl l ⁇ C(O)NRl0- and C1-C20 alkyl, and d) C1-C6 alkyl substituted with an unsubstituted or substituted group selected from aryl, heterocycle and C3- ClO cycloalkyl;
• R4 and R5 are independendy selected from: a) hydrogen, and b)
• R7 is independendy selected from: a) hydrogen, b) C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, Cl-C6 perfluoroalkyl, F, Cl, RlOO-, R!0C(O)NR10-, CN, N ⁇ 2, (RlO)2N-C(NRl )-, RlOC(O)-, RlO ⁇ C(O)-, -N(Rl )2, or Rl lOC(O)NRl -, and c) C1-C6 alkyl substituted by C1-C6 perfluoroalkyl, Rl°0-, Rl0C(O)NRl0-, (RlO) 2 N-C(NRl )-, RlOc(O)-,
• R8 is selected from: a) hydrogen, b) C2-C6 alkenyl, C2-C6 alkynyl, C l -C6 perfluoroalkyl, F, Cl,
• RlO is independently selected from hydrogen, -C6 alkyl, benzyl and aryl;
• Rl 1 is independently selected from Cl-C6 alkyl and aryl
• V is selected from: a) hydrogen, b) heterocycle selected from pyrrolidinyl, imidazolyl, pyridinyl, thiazolyl, pyridonyl, 2-oxopiperidinyl, indolyl, quinolinyl, isoquinolinyl, and thienyl, c) aryl, d) C1-C20 alkyl wherein from 0 to 4 carbon atoms are replaced with a a heteroatom selected from O, S, and N, and e) C2-C20 alkenyl, and provided that V is not hydrogen if Al is S(0)m and V is not hydrogen if Al is a bond, n is 0 and A is S(0)m;
• W is a heterocycle selected from pyrrolidinyl, imidazolyl, pyridinyl, thiazolyl, pyridonyl, 2-oxopiperidinyl, indolyl, quinolinyl, or isoquinolinyl;
• Z is independently H2 or O
• n 0, 1, 2, 3 or 4
• p 0, 1, 2, 3 or 4
• r 0 to 5, provided that r is 0 when V is hydrogen; and u is O or l;
• Rla is independendy selected from: hydrogen or C1-C6 alkyl
• Rib is independendy selected from: a) hydrogen, b) aryl, heterocycle, cycloalkyl, R OO-, -N(RlO)2 or alkenyl, c) C1-C6 alkyl unsubstituted or substituted by aryl, heterocycle, cycloalkyl, alkenyl, RlOO-, or -N(R10)2;
• R2a is selected from: a) hydrogen, b) C1-C6 alkyl unsubstituted or substituted by alkenyl, RlOO-, RHS(0) m -, Rl°C(O)NRl0-, CN, N3, (RlO)2N-C(NRlO)-, RlOC(O)-, RlO ⁇ C(O)-, -N(Rl )2, or RHOC(O)NR10-, c) aryl, heterocycle, cycloalkyl, alkenyl, Rl ⁇ O-,
• Rl lS(0)nr R 10 C(O)NRl0-, CN, NO2, (Rl°)2N- C(NRlO)-, RlOC(O)-, RlO ⁇ C(O)-, N3, -N(RlO) 2 , or Rl lOC(O)NRl0-, and d) C1-C6 alkyl substituted with an unsubstituted or substituted group selected from aryl, heterocyclic and
• R2b is hydrogen
• R3a and R3b are independently selected from: a) a side chain of a naturally occurring amino acid, b) an oxidized form of a side chain of a naturally occurring amino acid which is: i) methionine sulfoxide, or ii) methionine sulfone, c) substituted or unsubstituted C 1 -C 10 alkyl, C2-C 10 alkenyl, C3-C10 cycloalkyl, aryl or heterocyclic group, wherein the substituent is selected from F, Cl, Br, N02, Rl°0-, Rl lS(0) ⁇ r, R1°C(0)NR10-, CN, (RlO) 2 N-C(NRlO)., RlOC(O)-, RlO ⁇ C(O)-, N3, -N(RlO)2, Rl l ⁇ C(O)NRl - and Ci-Qo alkyl, and d) C1-C6 alkyl substituted with
• R4 and R5 are independendy selected from: a) hydrogen, and
• R7 is independendy selected from: a) hydrogen, b) C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 perfluoroalkyl, F, Cl, RlOO-, R10C(O)NR10-, CN, N02, (RlO)2N-C(NRlO)-, RlOc(O)-, RlO ⁇ C(O)-, -N(RlO)2, or RllOC(O)NRl0-, and c) C1-C6 alkyl substituted by C1-C6 perfluoroalkyl, Rl°0-, Rl C(O)NRl0-, (RlO) 2 N-C(NRlO)-, RlOC(O)-, RlO ⁇ C(O)-, -N(RlO)2, or RHOC(O)NR1 - ; R8 is selected from: a) hydrogen, b) C2-
• RlO is independendy selected from hydrogen, C1-C6 alkyl, benzyl and aryl;
• RU is independently selected from C1-C6 alkyl and aryl
• Rl2 is independently selected from hydrogen and C1-C6 alkyl
• Rl3 is independently selected from C1-C6 alkyl
• V is selected from: a) hydrogen, b) heterocycle selected from pyrrolidinyl, imidazolyl, pyridinyl, thiazolyl, pyridonyl, 2-oxopiperidinyl, indolyl, quinolinyl, isoquinolinyl, and thienyl, c) aryl, d) C1-C2O alkyl wherein from 0 to 4 carbon atoms are replaced with a a heteroatom selected from O, S, and N, and e) C2-C2O alkenyl, and provided that V is not hydrogen if A 1 is S(0)m and V is not hydrogen if Al is a bond, n is 0 and A is S(0) m ;
• W is a heterocycle selected from pyrrolidinyl, imidazolyl, pyridinyl, thiazolyl, pyridonyl, 2-oxopiperidinyl, indolyl, quinolinyl, or isoquinolinyl;
• Z is independently H2 or O
• Rla is independendy selected from: hydrogen or Cl-C6 alkyl
• Rib is independendy selected from: a) hydrogen, b) aryl, heterocycle, cycloalkyl, Rl ⁇ O-, -N(R10)2 or alkenyl, c) C1-C6 alkyl unsubstituted or substituted by aryl, heterocycle, cycloalkyl, alkenyl, Rl ⁇ O-, or -N(RlO)2;
• R2a is selected from: a) hydrogen, b) C 1 -C6 alkyl unsubstituted or substituted by alkenyl, R 1 ⁇ 0-, RllS(0) m -, Rl0C(O)NRl ., CN, N3, (RlO) 2 N-C(NRlO)., Rl C(O)-, Rl ⁇ C(O)-, -N(Rl )2, or RHOC(O)NR1 -, c) aryl, heterocycle, cycloalkyl, alkenyl, RlOO-, Rl lS(0) m -, R! C(O)NR1 -, CN, NO2, (Rl°)2N-
• R2b is hydrogen
• R4 and R5 are independendy selected from: a) hydrogen, and
• R7 is independendy selected from: a) hydrogen, b) C 1 -C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C 1 -C6 perfluoroalkyl, F, Cl, RlOO-, R! C(O)NR1 -, CN, N02, (RlO)2N-C(NRlO)-, RlOC(O)-, RlO ⁇ C(O)-, -N(Rl )2, or Rl lOC(O)NRl0-, and c) C 1 -C6 alkyl substituted by C 1 -C6 perfluoroalkyl, R 1 OO-,
• R8 is selected from: a) hydrogen, b) C2-C6 alkenyl, C2-C6 alkynyl, C 1 -C6 perfluoroalkyl, F, Cl, Rl O-, Rl lS(0)m-, R 10 C(O)NRl0-, CN, N ⁇ 2,
• RlO is independendy selected from hydrogen, C1-C6 alkyl, benzyl and aryl;
• Rl 1 is independently selected from C1-C6 alkyl and aryl
• V is selected from: a) hydrogen, b) heterocycle selected from pyrrolidinyl, imidazolyl, pyridinyl, thiazolyl, pyridonyl, 2-oxopiperidinyl, indolyl, quinolinyl, isoquinolinyl, and thienyl, c) aryl, d) C1-C2O alkyl wherein from 0 to 4 carbon atoms are replaced with a a heteroatom selected from O, S, and N, and e) C2-C20 alkenyl, and provided that V is not hydrogen if A 1 is S(0)m and V is not hydrogen if Al is a bond, n is 0 and A2 is S(0)m", W is a heterocycle selected from pyrrolidinyl, imidazolyl, pyridinyl, thiazolyl, pyridonyl, 2-oxopiperidinyl, indolyl, quinolinyl, or is
• Z is independendy H2 or O
• Rla is independently selected from: hydrogen or Cl-C6 alkyl
• R b is independendy selected from: a) hydrogen, b) aryl, heterocycle, cycloalkyl, Rl ⁇ O-, -N(R10)2 or alkenyl, c) C1-C6 alkyl unsubstituted or substituted by aryl, heterocycle, cycloalkyl, alkenyl, Rl ⁇ O-, or -N(RlO)2;
• R2a is selected from: a) hydrogen, b) C l -C6 alkyl unsubstituted or substituted by alkenyl, R 10 ⁇ -, Rl lS(0)m-, Rl°C(O)NRl0-, CN, N3, (R10)2N-C(NR10)-,
• R2b is hydrogen
• R4 and R5 are independendy selected from: a) hydrogen, and
• R7 is independently selected from: a) hydrogen, b) C l -C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C 1 -C6 perfluoroalkyl, F, Cl, RlOO-, R10C(0)NR10-, CN, N ⁇ 2,
• R8 is selected from: a) hydrogen, b) C2-C6 alkenyl, C2-C6 alkynyl, C 1-C6 perfluoroalkyl, F, Cl, RIOO-, Rl lS(0)m-, R1°C(0)NR10-, CN, N ⁇ 2, (RlO)2N-C(NRlO)-, RlOC(O)-, RlO ⁇ C(O)-, -N(RlO)2, or RllOC(O)NRl0-, and c) C l -C6 alkyl unsubstituted or substituted by C l -C6 perfluoroalkyl, F, Cl, RlOO-, RHS(0) m -, R1°C(0)NR10-, CN, (RlO)2N-C(NRlO)-, RlOc(O)-, RlO ⁇ C(O)-, -N(RlO)2, or RH ⁇ C(O)NRl
• RlO is independendy selected from hydrogen, C1-C alkyl, benzyl and aryl;
• Rl 1 is independendy selected from Cl-C6 alkyl and aryl
• V is selected from: a) hydrogen, b) heterocycle selected from pyrrolidinyl, imidazolyl, pyridinyl, thiazolyl, pyridonyl, 2-oxopiperidinyl, indolyl, quinolinyl, isoquinolinyl, and thienyl, c) aryl, d) C1-C20 alkyl wherein from 0 to 4 carbon atoms are replaced with a a heteroatom selected from O, S, and N, and e) C2-C2O alkenyl, and provided that V is not hydrogen if Al is S(0)m and V is not hydrogen if A is a bond, n is 0 and A2 is S(0) m ;
• W is a heterocycle selected from pyrrolidinyl, imidazolyl, pyridinyl, thiazolyl, pyridonyl, 2-oxopiperidinyl, indolyl, quinolinyl, or isoquinolinyl;
• Z is independendy H2 or O;
• amino acids which are disclosed are identified both by conventional 3 letter and single letter abbreviations as indicated below:
• alkyl is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms.
• cycloalkyl is intended to include non- 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 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, famesyl, geranyl, geranylgeranyl and the like.
• 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.
• aryl groups include phenyl, naphthyl, anthracenyl, biphenyl, tetrahydronaphthyl, indanyl, phenanthrenyl and the like.
• heterocycle or heterocyclic 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 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.
• heterocyclic elements include, but are not limited to, azepinyl, benzimidazolyl, benzisoxazolyl, benzofurazanyl, benzopyranyl, benzothiopyranyl, benzofuryl, benzothiazolyl, benzothienyl, benzoxazolyl, chromanyl, cinnolinyl, dihydrobenzofuryl, dihydro- benzothienyl, dihydrobenzothiopyranyl, dihydrobenzothio-pyranyl sulfone, furyl, irnidazolidinyl, imidazolinyl, imidazolyl, indolinyl, indolyl, isochromanyl, isoindolinyl, isoquinolinyl, isothiazolidinyl, isothiazolyl, isothiazolidinyl, mo ⁇ holinyl, naphthyridinyl,
• substituted aryl As used herein, the terms “substituted aryl”, “substituted heterocycle” and “substituted cycloalkyl” are intended to include the cyclic group which is substituted with 1 or 2 substitutents selected from the group which includes but is not limited to F, Cl, Br, CF3, NH2, N(Ci-C6 alkyl)2, N02, CN, (C1-C6 alkyl)0-, -OH, (C1-C6 alkyl)S(0) m -, (C1-C6 alkyl)C(0)NH-, H2N-C(NH)-, (C1-C6 alkyl)C(O)-, (C1-C6 alkyl)OC(O)-, N3,(Ci-C6 alkyl)OC(0)NH- and C1-C2O alkyl.
• cyclic moieties include, but are not limited to:
• cyclic moieties may optionally include a heteroatom(s).
• heteroatom-containing cyclic moieties include, but are not limited to:
• the pharmaceutically acceptable salts of the compounds of this invention include the conventional non-toxic salts of the compounds of this invention as formed, e.g., from non-toxic inorganic or organic acids.
• 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.
• any substituent or variable e.g., RlO, Z, n, etc.
• -N(RlO)2 represents -NHH, -NHCH3, -NHC2H5, etc.
• 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 pharmaceutically acceptable salts of the compounds of this invention can be synthesized from the compounds of this invention which contain a basic moiety by conventional chemical methods. Generally, the salts are prepared by reacting the free base with stoichiometric amounts or with an excess of the desired salt-fo ⁇ xiing 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 Bodanszky 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 Stewart et al., “Solid Phase Peptide Synthesis", Second Edition, Pierce Chemical Company, 1984. The teachings of these works are hereby inco ⁇ orated by reference.
• Reaction B Preparation of a reduced peptide subunit by reductive alkylation of an amine by an aldehyde using sodium cyanoborohydride or other reducing agents.
• Reaction Schemes E - M illustrate reactions wherein the non-sulfliydryl-containing moiety at the N-terminus of the compounds of the instant invention is attached to an aminomethylbenzamide subunit which may be further elaborated to provide the instant compounds.
• These reactions may be employed in a linear sequence to provide the compounds of the invention or they may be used to synthesize fragments which are subsequendy joined by the reactions described in Reaction Schemes A - D.
• Schemes A - D can be reductively alkylated with a variety of aldehydes, such as I, as shown in Reaction Scheme E.
• the aldehydes can be prepared by standard procedures, such as that described by O. P. Goel, U. Krolls, M. Stier and S. Kesten in Organic Syntheses. 1988, 67, 69-75, from the appropriate amino acid (Reaction Scheme E).
• the reductive alkylation can be accomplished at pH 5-7 with a variety of reducing agents, such as sodium triacetoxyborohydride or sodium cyanoborohydride in a solvent such as dichloroethane, methanol or dimethylformamide.
• the product II can be deprotected to give the final compounds HI with trifluoroacetic acid in methylene chloride.
• the final product UI is isolated in the salt form, for example, as a trifluoroacetate, hydrochloride or acetate salt, among others.
• the product diamine UI can further be selectively protected to obtain IV, which can subsequendy be reductively alkylated with a second aldehyde to obtain V. Removal of the protecting group, and conversion to cyclized products such as the dihydroimidazole VII can be accomplished by literature procedures.
• the aminomethylbenzamide subunit can be reductively alkylated with other aldehydes such as l-trityl-4- carboxaldehyde or l-trityl-4-imidazolylacetaldehyde, to give products such as VDI (Reaction Scheme F).
• the trityl protecting group can be removed from VHI to give IX, or alternatively, VHI can first be treated with an alkyl halide then subsequendy deprotected to give the alkylated imidazole X.
• the aminomemylbenzamide subunit can be acylated or sulfonylated by standard techniques.
• the imidazole acetic acid XI can be converted to the acetate Xm by standard procedures, and Xlll can be first reacted with an alkyl halide, then treated with refluxing methanol to provide the regiospecifically alkylated imidazole acetic acid ester XTV.
• Hydrolysis and reaction with the aminomethylbenzamide subunit in die presence of condensing reagents such as l-(3-dimethylaminopropyl)-3- ethylcarbodiimide (EDC) leads to acylated products such as XV.
• the aminomethylbenzamide subunit is reductively alkylated with an aldehyde which also has a protected hydroxyl group, such as XVI in Reaction Scheme H
• the protecting groups can be subsequendy removed to unmask the hydroxyl group (Reaction Schemes H, I).
• the alcohol can be oxidized under standard conditions to e.g. an aldehyde, which can then be reacted with a variety of organometallic reagents such as Grignard reagents, to obtain secondary alcohols such as XX.
• the fully deprotected amino alcohol XXI can be reductively alkylated (under conditions described previously) with a variety of aldehydes to obtain secondary amines, such as XXII (Reaction Scheme J), or tertiary amines.
• the Boc protected amino alcohol XVHI can also be utilized to synthesize 2-aziridinylmethylpiperazines such as XXJULl (Reaction Scheme K). Treating XVHI with lj'-sulfonyldiimidazole and sodium hydride in a solvent such as dimethylfo ⁇ namide led to the formation of aziridine XXHI . The aziridine reacted in the presence of a nucleophile, such as a thiol, in the presence of base to yield the ring-opened product XXTV .
• a nucleophile such as a thiol
• aminomethylbenzamide subunit can be reacted with aldehydes derived from amino acids such as O-alkylated tyrosines, according to standard procedures, to obtain compounds such as XXX, as shown in Reaction Scheme L.
• R 1 is an aryl group
• XXX can first be hydrogenated to unmask the phenol, and the amine group deprotected with acid to produce XXXI.
• the amine protecting group in XXX can be removed, and O-alkylated phenolic amines such as XXXII produced.
• Reaction Scheme M illustrates a one pot synthesis of an instant compound wherein the N-terminus nitrogen is substituted with two different non-sulfhydryl-containing moieties.
• the aminomethylbenzamide subunit is treated with one equivalent of an appropriate aldehydea and, after the reductive adduct has been formed, the in situ intermediate is treated with an equivalent of a different aldehyde.
• the -NR 4 R5 moiety of the compounds of the instant invention may provide advantages over a cysteinyl moiety that is incorporated in other types of molecules that are known to be inhibitors of famesyl protein transferase.
• modification of the benzodiazepine compounds described in published PCT application WO 94/26723 with the such -NR 4 R 5 substituents as described herein will provide inhibitors of famesyl protein transferase of the following formulae A and B:
• R, R' and R 25 are as defined in WO 94/26723
• R4benz, R4'benz R7benz and Wbenz are R 4 , R4', R7 and W respectively as defined in WO 94/26723 and R a and Rb are defined as R 4 and R5 are defined herein respectively.
• the following combinations of R a and R- are selected for incorporation into the compounds of formulae A and B:
• benzodiazepine compound would be selected from the following formulae:
• R and R * are as defined in WO 94/26723 and W'benz i s r as defined in WO 94/26723 and Ra and Rb are defined as R 4 and R- are defined herein respectively.
• Such benzodiazepine analogs may be synthesized by techniques well known in the art, as well as procedures outlined in WO 94/26723. General methods of synthesis of the benzediazapine analogs of this invention are shown in Schemes N, P and Q. Typically a convergent route is employed, which joins the key intermediate 9 (Scheme N) with suitably functionalized amine and R a and Rb components (Schemes P and Q) using standard amide bond-forming procedures.
• the protected amino acid 9 may be prepared from a suitably substituted 2-aminobenzophenone (1).
• Many 2- aminobenzophenones are known in the art or are available form commercial sources such as Aldrich Chemical Co. General methods for the synthesis of new 2-aminobenzophenones may be found in the literature (c.f. Walsh, D. A. Synthesis, 677-688 (1980).
• Alkylation of 3 with a substituted organic ester (4), preferably tert-butyl bromacetate, in the presence of a base, preferably CS2CO3 in l-methyl-2- pyrrolidinone at ambient temperature gives 5.
• 3 may be alkylated at N-l with a variety of other alkylating agents, for instance, esters of substituted or unsubstituted acrylates, 4-bromobutanoates, etc.
• Branched compounds i.e.
• R 4 benz and/or R4"benz ⁇ H) S ma y e prepared by generation of the polyanion of 5 with base and alkylation with an appropriate alkyl halide. Subsequent to alkylation, the ester of 5 may be cleaved with an acid such as TFA (for the tert-butyl esters) or under mild aqueous base hydrolysis (for other alkyl esters) at temperatures between 0 and 25 °C.
• TFA for the tert-butyl esters
• mild aqueous base hydrolysis for other alkyl esters
• the acid 6 is converted to amino acid 8 via reaction of the dianion, generated with at least two equivalents of a strong base with an electrophilic animating agent.
• 6 may be halogenated and reacted with an amine source such as azide (followed by reduction) or ammonia.
• 6 is reacted with 4 equivalents of potassium tert- butoxide in glyme at -5 °C for 30 min and treated with 1.1 equivalents of isobutyl nitrite.
• the resulting oxime 7 can then be reduced to the racemic amino acid 8 using a variety of reductants, preferably hydrogenation at 40 psig in the presence of Ruthenium on carbon or Raney nickel in methanol at 50 to 70 ⁇ C for 1-4 days.
• reductants preferably hydrogenation at 40 psig in the presence of Ruthenium on carbon or Raney nickel in methanol at 50 to 70 ⁇ C for 1-4 days.
• Amino acid 8 is then suitably protected for selective coupling at the carboxyl terminus.
• 8 can be converted to the N-BOC derivative 9 using standard amino acid protection conditions, preferably, reaction with equimolar amounts of di-tert-butyl dicarbonate and triethyl amine in DMF/ water at ambient temperature.
• R a ⁇ H 9 can be alkylated at nitrogen with a wide variety of alkylating agents including n-alkyl, branched alkyl, and benzyl, according to the standard procedure of Benoiton, et al., Can.
• 3 may be directly alkylated with the "top" sidechain in one intact piece, as shown in Scheme Q.
• P.G. is a suitably selected protecting group which is utilized if necessary.
• the compounds of this invention inhibit Ras famesyl 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 pharmaceutical agents for mammals, especially for humans. These compounds may be administered 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 administered to mammals, preferably humans, either alone or, preferably, in combination with pharmaceutically acceptable carriers or diluents, optionally with known adjuvants, such as alum, in a pharmaceutical composition, according to standard pharmaceutical practice.
• the compounds can be administered orally or parenterally, including the intravenous, intramuscular, intraperitoneal, subcutaneous, rectal and topical routes of administration.
• the selected compound may be administered, for example, in the form of tablets or capsules, or as an aqueous solution or suspension.
• carriers which are commonly used include lactose and com starch, and lubricating agents, such as magnesium stearate, are commonly added.
• useful diluents include lactose and dried com starch.
• 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.
• sterile solutions of the active ingredient are usually prepared, and the pH of the solutions should be suitably adjusted and buffered.
• the total concentration of solutes should be controlled in order to render the preparation isotonic.
• the present invention also encompasses a pharmaceutical composition useful in the treatment of cancer, comprising the ad ⁇ iinistration of a therapeutically effective amount of the compounds of this invention, with or without pharmaceutically acceptable carriers or diluents.
• Suitable compositions of this invention include aqueous solutions comprising compounds of this invention and pharmacologically acceptable carriers, e.g., saline, at a pH level, e.g., 7.4.
• the solutions may be introduced into a patient's intramuscular blood-stream by local bolus injection.
• 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.
• a suitable amount of compound is administered to a mammal undergoing treatment for cancer.
• Administration occurs in an amount between about 0.1 mg/kg 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/kg of body weight per day.
• the compounds of the instant invention are also useful as a component in an assay to rapidly determine the presence and quantity of famesyl-protein transferase (FPTase) in a composition.
• FPTase famesyl-protein transferase
• the composition to be tested may be divided and the two portions contacted with mixtures which comprise a known substrate of FPTase (for example a tetrapeptide having a cysteine at the amine terminus) and famesyl pyrophosphate and, in one of the mixtures, a compound of the instant invention.
• the chemical content of the assay mixtures may be determined by well known immunological, radiochemical or chromatographic techniques. Because the compounds of the instant invention are selective inhibitors of FPTase, absence or quantitative reduction of the amount of substrate in the assay mixture without the compound of the instant invention relative to the presence of the unchanged substrate in the assay containing the instant compound is indicative of the presence of FPTase in the composition to be tested.
• potent inhibitor compounds of the instant invention may be used in an active site titration assay to determine the quantity of enzyme in the sample.
• a series of samples composed of aliquots of a tissue extract containing an unknown amount of famesyl-protein transferase, an excess amount of a known substrate of FPTase (for example a tetrapeptide having a cysteine at the amine terminus) and famesyl pyrophosphate are incubated for an appropriate period of time in the presence of varying concentrations of a compound of the instant invention.
• concentration of a sufficientiy potent inhibitor i.e., one that has a Ki substantially smaller than the concentration of enzyme in the assay vessel
• concentration of a sufficientiy potent inhibitor i.e., one that has a Ki substantially smaller than the concentration of enzyme in the assay vessel
• Step A Preparation of N-( 1 (S)-carbomethoxy-3-methylthio ⁇ ro ⁇ yl)-
• Step B Preparation of N-( 1 (S)-carbomethoxy-3-methylthiopropyl)- 3-azidomethylbenzamide To a stirred solution of the product from Step A (13.52 g,
• Step D Preparation of N-( 1 (S)-carbomethoxy-3-methylthiopropyl) 3- [( 1 -triphenylmethyl)-4-imidazolylmethyl]aminomethyl benzamide
• the ethyl acetate layer was washed with 125 mL each of saturated sodium hydrogen carbonate and saturated sodium chloride, dried over magnesium sulfate, and concentrated in vacuo to yield 0.363 g of cmde product.
• the cmde product was chromatographed on silica gel eluting with chloroform/methanol 95/5 to afford the tide compound.
• Step E Preparation of N-( 1 (S)-carbomethoxy-3-methylthiopropyl)- 3-(4-in idazolylmemyl)am omemylbenzamide dihvdrochloride
• Step F Preparation of N-( 1 (S)-carboxy-3-methylthiopropyl)-3-(4- imidazolylmethvDaminomethyl benzamide dihydrochloride
• Step E The product from Step E (0.030 g, 0.067mmol) was dissolved in 5 mL of methanol and 3 mL of 5% sodium hydroxide and stirred for 1 h under nitrogen.
• the reaction mixture was injected directiy onto a preparative reverse phase HPLC column with conditions identical as in the preparation of the compound in Step E. Pure fractions were pooled, evaporated in vacuo, and the sample was converted to the hydrochloride salt as before. Lyophillization overnight afforded 0.022 g (0.051 mmol) of the tide compound as a solid.
• Step A Preparation of N-( 1 (S)-carbomethoxy-3-methylthiopropyl)- 3- [N,N--7w[( 1 -triphenylmethyl)-4-imidazolylmethyl]- aminomethyl-benzamide
• Step C To a solution of the product from Example 1 , Step C (0.100 g, 0.357 mmol) in 10 mL of 1,2-dichloroethane was added glacial acetic acid dropwise until the pH was 5.5.
• Step B Preparation of N-( 1 (S)-carbomethoxy-3-methylthiopropyl)- 3-[N,N-fei_;(4-imidazolylmemyl)_u nomethyl]benzamide dihvdrochloride
• triethylsilane 0.159 g, 1.36 mmol
• trifluoroacetic acid 5 mL
• the 0.1% TFA/water:methanol solution was injected directiy onto a Delta-Pak (C-18, lOOA, 15 mm, 40mm x 100mm) preparative HPLC column.
• the gradient at 40 mL min was 100% 0.1% TFA/water for 5 min followed by 95% 0.1% TFA/water to 60% 0.1% TFA/water :40% 0.1% TFA/acetonitrile over 40 min.
• the pure fractions were pooled, evaporated to near dryness, and then taken up in 5 mL of water.
• the aqueous solution was passed through a 1.2 gm. column of Bio-Rad AG 3-X4 chloride anion exchange resin.
• Step C Preparation of N-( 1 (S)-carboxy-3-methylthiopropyl)-3- [N,N-_?i5(4-iinidazolemethyl)an_Luiomethyl]benzamide dihydrochloride
• the compound from Step B (0.035 g, 0.052 mmol) was dissolved in 5 mL of methanol and 3 mL of 5% sodium hydroxide and stirred for 1 hr under nitrogen.
• the reaction mixture was injected directly onto a preparative HPLC column with conditions identical as in Step B. Pure fractions were pooled, evaporated, and die sample converted to the hydrochloride salt as before. Lyophilization overnight afforded the title compound as a solid.
• Step A Preparation of Methyl 3-chloromethyl benzoate To a solution of triethylamine ( 11.0 mL) in methanol (150 mL) at 0°C was added 3-chloromethylbenzoyl chloride (5.0 g) dropwise. After stirring at 20° C for 0.5 h the solution was concentrated in vacuo. The residue was partitioned with 125 mL of water and 150 mL of ethyl acetate. The ethyl acetate layer was washed with 125 mL each of saturated sodium hydrogen carbonate, 2% potassium hydrogen sulfate and saturated sodium chloride, dried over magnesium sulfate, and concentrated in vacuo to yield the title compound.
• Step B of Example 1 was used to prepare the tide compound.
• Step C of Example 1 was used to prepare the title compound.
• Step F Preparation of 3- [(t-butyloxycarbonyl)-N-methyl- aminomethyll benzoic acid
• Step G Preparation of N-( 1 (S)-carbome ⁇ oxy-3-methylthiopropyl)- 3-r(t-but ⁇ loxycarbonyl)-N-methylammomethyl1benzamide
• hydroxybenzotriazole (0.16 g)
• EDC (0.19 g)
• N-methylmorpholine (0.40 mL)
• (S) methione methyl ester hydrochloride (0.203 mg). After stirring for 2 h the solution was concentrated in vacuo and the residue was partitioned with water and ethyl acetate.
• Step H Preparation of N-( 1 (S)-carbomethoxy-3-methylthiopropyl)- 3-(N-methylaminomethyl)benzamide trifluoroacetate
• trifluoroacetic acid 33% by volume
• Step I Preparation of N-( 1 (S)-carbomethoxy-3-methylthiopropyl)- 3-[(l-triphenylmethyl)-4-imidazolylmethyl-N-methyl- aminomethyllbenzamide
• Step D of Example 1 was used to prepare the title compound.
• Step J Preparation of N-( 1 (S)-carbomethoxy-3-methylthiopropyl)- 3- [(4-imidazolylmethyl) -N-methyl-minomethyl]benzamide dihydrochloride Starting with the compound from Step I (0.24 g) the method described in Step E of Example 1 was used to prepare the tide compound. FAB mas spectrum m/e 391 (m+1). Analysis for C19H26N4O3S • 5.0 HCl • 0.5 H2O: Calculated: C, 39.32; H, 5.56; N, 9.65; Found: C, 39.33; H, 5.57; N, 9.38.
• Step K Preparation of N-( 1 (S)-carboxy-3-methylthio ⁇ ro ⁇ yl)-3-[(4- imidazolememyl) -N-memyl-aminomethyl]benzamide dihvdrochloride
• Step F of Example 1 Starting with the compound from Step J (0.035 g) the method described in Step F of Example 1 was used to prepare the above title compound. FAB mas spectmm m/e 377 (m+1). Analysis for C18H24N4O3S • 3.70 HCl • 0.2 H2O: Calculated C, 42.05; H, 5.51; N, 10.90; Found: C, 42.09; H, 5.49; N, 10.70.
• Step B Preparation of N-( 1 (S)-carbomethoxy-3-methylthiopropyl)-
• Step D Preparation of N-(l (S)-carbomethoxy-3-methylthiopropyl)- 4- [( 1 -triphenylmethyl)-4-imidazolylmethyl] - aminobenzamide
• the method described in Step D of Example 1 was used to prepare the title compound.
• Step E Preparation of N-( 1 (S)-carbomethoxy-3-methylthiopropyl)- 4-(4-imidazolylmethyl)aminobenzamide dihvdrochloride
• Step F Preparation of N-( 1 (S)-carboxy-3-methylthiopropyl)-4- [(4- imidazolylmethv aminolbenzamide dihvdrochloride
• Step F of Example 1 the method described in Step F of Example 1 was used to prepare the title compound.
• Example 5 Using the appropriate starting materials the methods described above for Example 4 were used to prepare Examples 5-7.
• Step A N-(l (S)-Carbomethoxy-3-methylthiopropyl)-3-[N-(4- imidazolylymethyl)-N-(4-nitrobenzyl)aminomethyl]- benzamide ditrifluoroacetate
• N-( 1 (S)-carbomethoxy-3-methylthiopropyl)-3- aminomethylbenzamide (0.104 g, 0.352 mmol) was dissolved in dichloroethane (5 mL). Cmshed molecular sieves ( 0.209 g) and sodium triacetoxyborohydride (0.186 g, 0.881 mmol). The pH was about 7.5. 4- Nitrobenzaldehyde (0.0533 g, 0.352 mmol) was added plus 0.5 drop of acetic acid to bring the pH to about 7. The reaction was stirred 2 h under nitrogen at 20°C.
• the cmde product was chromatographed on silica gel with 50% ethyl acetate in hexane. This chromatographed product was dissolved in dichloromethane ( 7 mL); triethylsilane (0.5 mL, 3.13 mmol) was added and then trifluoroacetic acid ( 3.5 mL). After 0.5 h at 20°C, the solvent was evaporated and the residue partitioned between hexane and water.
• the aqueous solution was purified by preparative reverse phase HPLC using a 100 mm Waters PrepPak® reverse phase column (DeltaPakTM C18, 50 ⁇ M, 100 A) and pure product isolated by gradient elution using 80% 0.1% trifluoroacetic acid in water (Solvent A) and 20% 0.1% trifluoroacetic acid in acetonitrile (Solvent B) to 55% Solvent A and 45% Solvent B.
• the pure fractions were combined and the solvent evaporated, and the pure product was dissolved in water and lyophilized to give the title compound as a clear, pale yellow solid.
• Step B Preparation of N-(l(S)-carboxy-3-methylthiopro ⁇ yl)-3-[N- (4-imidazolylymemyl)-N-(4-mtrobenzyl)aminomethyl]- benzamide ditrifluoroacetate
• Step A The product from Step A ( 0.045 g, 0.0608 mmol) was dissolved in methanol (4 mL) and 0.5 mL of 10% NaOH solution was added to take pH to about 12. Water (4 mL) was added. At 3 h reaction was purified and lyophilized according to the procedure described in Step A to the tide compound as a white solid.
• Step A Preparation of N-( 1 (S)-carboxyme yl-3-methylthiopropyl)- 3-rN.N-frfo-(4-mtrophenylmethyl)l ditrifluoroacetate
• the product from Example 1, Step 3 (0.100 g, 0.337 mmol) was dissolved in dichloroethane (5 mL).
• p-Nitrobenzaldehyde, sodium triacetoxyborohydride (0.214 g, 1.01 mmol) and cmshed molecular sieves were added, and the pH adjusted to 5.5 with acetic acid and triethylamine.
• the reaction was stirred at 20°C overnight, quenched with saturated sodium bicarbonate, and partitioned between ethyl acetate and saturated sodium bicarbonate.
• the organic phase was washed with 2% potassium hydrogen sulfate, saturated sodium bicarbonate, saturated brine, and dried over magnesium sulfate.
• the cmde product was purified by silica gel chromatography using 40% ethyl acetate in hexane. This product was further purified by preparative reverse phase HPLC using a gradient elution from 85% water, 15% acetonitrile to 20% water over a period of 40 min. (solvents contained 0.1% trifluoroacetic acid).
• Bovine FPTase was assayed in a volume of 100 ⁇ l containing 100 mM _V-(2-hydroxy ethyl) piperazine-W-(2-ethane sulfonic acid) (HEPES), pH 7.4, 5 mM MgCl2, 5 mM dithiothreitol (DTT), 100 mM [3H]-famesyl diphosphate ([3H]-FPP; 740 CBq/mmol, New England 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]-FPP was utilized during the reaction period.
• Purified compounds were dissolved in 100% dimethyl sulfoxide (DMSO) and were diluted 20-fold into the assay. Percentage inhibition is measured by the amount of incorporation of radioactivity in the presence of the test compound when compared to the amount of incorporation in the absence of the test compound.
• DMSO dimethyl sulfoxide
• 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 ⁇ M ZnCl 2 and 100 ⁇ M Ras-CVIM were added to the reaction mixture. Reactions were performed for 30 min., stopped with 100 ⁇ l 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 human FPTase by the assay described above and were found to have IC50 of ⁇ 100 ⁇ M.
• the cell line used in this assay is a v-ras line derived from either Ratl or NIH3T3 cells, which expressed viral Ha-ras p21.
• the assay is performed essentially as described in DeClue, J.E. et al, Cancer Research 57:712-717, (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%).
• the cells After 4 hours at 37°C, the cells are labelled in 3 ml methionine-free DMEM supple-meted with 10% regular DMEM, 2% fetal bovine serum and 400 mCi[35S]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/l M DTT/10 mg/ml aprotinen/2 mg/ml leupeptin/2 mg/ml antipain/0.5 mM PMSF) and the lysates cleared by centrifugation at 100,000 x g for 45 min.
• 1 ml lysis buffer 1% NP40/20 mM HEPES, pH 7.5/5 mM MgCl2/l M DTT/10 mg/ml aprotinen/2 mg/ml leupeptin/2 mg/ml antipain/0.5 mM PMSF
• the immunoprecipitates are washed four times with IP buffer (20 nM HEPES, pH 7.5/1 mM EDTA/1% Triton X-100.0.5% deoxycholate/0.1%/SDS/0.1 M NaCl) boiled in SDS-PAGE sample buffer and loaded on 13% acrylamide gels. When the dye front reached the bottom, the gel is fixed, soaked in Enlightening, dried and autoradiographed. The intensities of the bands corresponding to farnesylated and nonfarnesylated ras proteins are compared to determine the percent inhibition of famesyl transfer to protein.
• IP buffer 20 nM HEPES, pH 7.5/1 mM EDTA/1% Triton X-100.0.5% deoxycholate/0.1%/SDS/0.1 M NaCl
• Rat 1 cells transformed with either v-ras, v-raf, or v-mos are seeded at a density of 1 x 10 4 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 semm) over a bottom agarose layer (0.6%). Both layers contain 0.1% methanol or an appropriate concentration of the instant compound (dissolved in methanol at 1000 times the final concentration used in the assay).
• the cells are fed twice weekly with 0.5 ml of medium A containing 0.1% methanol or the concentration of the instant compound. Photomicrographs are taken 16 days after the cultures are seeded and comparisons are made.

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