Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/06—Dipeptides
  • C07K5/06139—Dipeptides with the first amino acid being heterocyclic
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• Ras proteins are 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 signal 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.
• Mutated ras genes (Ha-ras, Ki4a-r_ «, Ki4b-r_._. and N-ras) are found in many human cancers, including colorectal carcinoma, exocrine pancreatic carcinoma, and myeloid leukemias. The protein products of these genes are defective in their GTPase activity and constitutively transmit a growth stimulatory signal.
• Ras must be localized to the plasma membrane for both normal and oncogenic functions. At least 3 post-translational modifications are involved with Ras membrane localization, and all 3 modifications occur at the C-terminus of Ras.
• the Ras C-terminus contains a sequence motif termed a "CAAX” or "Cys-Aaa 1 -Aaa--Xaa” box (Cys is cysTfcine, Aaa is an aliphatic amino acid, the Xaa is any amino acid) (Willumsen et al. Nature 310: ' ⁇ ' -S ⁇ ( 1984)).
• this motif serv es as a signal sequence for the enzymes famesyl-protein transferase or geranylgeranvl-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.
• famesylated 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 famesylated. James, et al., have also suggested that there are famesylated proteins of unknown structure and function in addition to those listed above.
• FPTase famesyl-protein transferase
• FPP famesyl diphosphate
• Ras protein substrates
• the peptide derived inhibitors that have been described are generally cysteine containing molecules that are related to the CAAX motif that is the signal for protein prenylation.
• 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 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.
• famesyl-protein transferase inhibitors are inhibitors of proliferation of vascular smooth muscle cells and are therefore useful in the prevention and therapy of arteriosclerosis and diabetic disturbance of blood vessels (JP H7-1 12930).
• an object of this invention to develop peptidomimetic compounds that do not have a thiol moiety, and that will inhibit famesyl-protein transferase and thus, the post-translational famesylation of proteins. It is a further object of this invention to develop chemotherapeutic compositions containing the compounds of this invention and methods for producing the compounds of this invention.
• the present invention comprises analogs of the CA A ⁇ X motif of the protein Ras that is modified by famesylation in vivo. These CA! A ⁇ X analogs inhibit the famesylprotein transferase. Furthermore, these CA! A ⁇ X 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 the instant invention also incorporate a cyclic amine moiety in the A ⁇ position of the motif. Further contained in this invention are chemotherapeutic compositions containing these famesyl transferase inhibitors and methods for their production.
• the compounds of this invention inhibit the famesyl-protein transferase.
• the famesyl-protein transferase inhibitors are illustrated bv the formula I: - A 1 (CR 1a 2 ) n A 2 (CR 1a 2 ) n
• Rla and Rib are independently selected from: a) hydrogen, b) aryl, heterocycle, cycloalkyl, alkenyl, alkynyl, R ⁇ O-, ⁇ S(0) m -, Rl0c(O)NRl0-, CN, N ⁇ 2, (R 10 )2N- C(NRl )-, RlOC(O)-, RlO ⁇ C(O)-, N3, -N(Rl )2, or Rl lOC(O)NRl -, c) C1-C alkyl unsubstituted or substituted by aryl, heterocyclic, cycloalkyl, alkenyl, alkynyl, Rl ⁇ O-,
• R2 and R3 are independently selected from: a) a side chain of a naturally occurring amino acid, b) an oxidized form of a side chain of a naturally occurring amino acid which is: i) methionine sulfoxide, or ii) methionine sulfone, and c) substituted or unsubstituted C l -C20 alkyl, C2-C2O alkenyl, C3-C10 cycloalkyl, aryl or heterocyclic group, wherein the substituent is selected from F, Cl, Br, N(Rl0)2, N02, R ⁇ O-, Rl l S(0) ⁇ r, R 10 C(O)NR l0-, CN, (RlO) 2 N-C(NRlO)-, R 10 C(O)-, R!OOC(0)-, N3,
• R2 and R3 are combined to form - (CH2)s - ;
• R2 or R3 are combined with R6 to form a ring such that
• R4a, R4b, R7a and R7b are independently selected from: a) hydrogen, b) C l -C6 alkyl unsubstituted or substituted by alkenyl, R OO-, R n S(0) m -, Rl0c(O)NRl0-, CN, N3, (R10)2N-C(NR10)-, RlOc(O)-, RlO ⁇ C(O)-, -N(RlO) 2 , or R 10C(0)NR10-, c) aryl, heterocycle, cycloalkyl, alkenyl, Rl ⁇ O-, R n S(0) m -, Rl0c(O)NRl0-, CN, N02, (Rl°)2N- C(NRlO)-, RlOC(O)-, RlO ⁇ C(O)-, N3, -N(RlO) 2 , or Rl lOC(O)NRl0-, and d)
• R5a and R5b 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 -C20 alkyl, C2-C2O alkenyl.
• C3-C10 cycloalkyl, aryl or heterocycle group wherein the substituent is selected from F, Cl, Br, (RlO)2NC(0)-, N ⁇ 2, R 10 O-, RHS(0) m -, Rl0C(O)NRl -, CN, (RlO)2N-C(NRl )-, RlOc(O)-, RlO ⁇ C(O)-, N3, -N(Rl )2, RHOC(O)NR10- and C1-C20 alkyl, d) C1-C6 alkyl substituted with an unsubstituted or substituted group selected from aryl, heterocycle and C3- C10 cycloalkyl; or
• R5a and R5b 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(CORlO)- ;
• R6 is independently selected from hydrogen or C1-C6 alkyl
• R8 is independently selected from: a) hydrogen, b) aryl, heterocycle, cycloalkyl, alkenyl, alkynyl, perfluoroalkyl, F, Cl, Br, RlOO-, R lS(0) m -, Rl0C(O)NRl0-, CN, N ⁇ 2 .
• R 10 2N-C(NRlO)-, RlOc(O)-,
• R9 is selected from: a) hydrogen, b) alkenyl, alkynyl, perfluoroalkyl, F. Cl, Br, R ! 0 O-,
• R O is independently selected from hydrogen, C1-C6 alkyl, benzyl and aryl;
• Rl is independently selected from Cl-C6 alkyl and aryl
• Q is a substituted or unsubstituted nitrogen-containing C4-C9 mono or bicychc ring system, wherein the non-nitrogen containing ring may be a C5-C7 saturated ring;
• V is selected from: a) hydrogen, b) heterocycle, c) aryl, d) C1-C20 alkyl wherein from 0 to 4 carbon atoms are replaced with a a heteroatom selected from O, S, and N, and e) C2-C20 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 A ⁇ is S(0)m;
• R2 and R3 are independently selected from: a) a side chain of a naturally occurring amino acid, b) an oxidized form of a side chain of a naturally occurring amino acid which is: i) methionine sulfoxide. or ii) methionine sulfone.
• R2 and R3 are combined to form - (CH2)s - ;
• R2 or R3 are combined with R6 to form a ring such that
• R4a, R4b, R7a and R7b are independently selected from: a) hydrogen, b) C 1 -C ⁇ alkyl unsubstituted or substituted by alkenyl, R 1 0 O-, Rl lS(0)m-, R10C(O)NR10-, CN, N3, (RlO)2N-C(NRlO)-,
• R a and R5b are independently selected from: a) a side chain of a naturallv occur ⁇ nc 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 C1-C20 alkyl, C2-C20 alkenyl,
• C1-C20 alkyl d) C1-C6 alkyl substituted with an unsubstituted or substituted group selected from aryl, heterocycle and C3- C 10 cycloalkyl; or
• R5a and R5b 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(CORlO)- ;
• R6 is independently selected from hydrogen or C1-C6 alkyl
• R8 is independently selected from: a) hydrogen, b) aryl, heterocycle, cycloalkyl, alkenyl, alkynyl, perfluoroalkyl, F, Cl, Br, RlOO-, Rl lS(0) m -,
• R9 is selected from: a) hydrogen, b) alkenyl, alkynyl, perfluoroalkyl, F, Cl, Br, R 0 ⁇ -, R n S(0) m -, R 10 C(O)NRl0-, CN, N02, (Rl°)2N-C- (NR )-, RlOc(O)-, RlO ⁇ C(O)-, N3, -N(Rl )2, or RllOC(O)NRl -, and c) C l -C6 alkyl unsubstituted or substituted by perfluoroalkyl, F, Cl, Br, Rl O-, Rl lS(0) m -, R1°C(0)NR10-, CN, (RlO) 2 N-C(NRlO)-, RlOc(O)-, RlOOC(O)-, N3, -N(Rl ) 2 , or Rl lOC(O)NRl0-
• RlO is independently selected from hydrogen, C1-C6 alkyl, benzyl and aryl;
• Rl 1 is independently selected from C1-C6 alkyl and aryl
• Rl2 is a) substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted C5-C8 cycloalkyl, or substituted or unsubstituted cyclic amine, wherein the substituted alkyl, cycloalkyl or cyclic amine is substituted with 1 or 2 substituents independently selected from:
• R 1 is independently selected from hydrogen and Cl -C6 alkyl
• Q is a substituted or unsubstituted nitrogen-containing C4-C9 mono or bicyclic ring system, wherein the non-nitrogen containing ring may be a C5-C7 saturated ring;
• V is selected from: a) hydrogen, b) heterocycle, c) aryl, d) C1 -C20 alkyl wherein from 0 to 4 carbon atoms are replaced with a a heteroatom selected from O, S, and N, and e) C2-C20 alkenyl, provided that V is not hydrogen if Al is S(0) 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-, R n S(0) m -, Rl c(O)NRl0-, CN, N ⁇ 2, (Rl°)2N- C(NRl )-, Rl0c(O)-, Rl ⁇ C(O)-, N3, -N(Rl ) 2 , or Rl lOC(O)NRl0-, c) C1-C6 alkyl unsubstituted or substituted by aryl, heterocyclic, cycloalkyl, alkenyl, alkynyl, Rl ⁇ O-, R ⁇ S(0) m -, RlOC(0)NRlO-, CN, (R10)2N-C(NR10)-, RlOC(O)-, RlO ⁇ C(O)-, N3, -N(Rl )2, or R
• R2 and R3 are independently selected from: a) a side chain of a naturally occurring amino acid, b) an oxidized form of a side chain of a naturally occurring amino acid which is: i) methionine sulfoxide, or ii) methionine sulfone, and c) substituted or unsubstituted C 1 -C20 alkyl, C2-C20 alkenyl, C3-C10 cycloalkyl, aryl or heterocyclic group,
• R 10 O-, R l l S(0)rn-.
• R 10 C(O)NR l0-,
• R2 and R3 are combined to form - (CH2)s - ;
• R2 or R3 are combined with R6 to form a ring such that
• R4a, R4b, R7a and R7b are independently selected from: a) hydrogen, b) C 1 -C6 alkyl unsubstituted or substituted by alkenyl, R 1 OO-, R n S(0) m -, R 10 C(O)NRl0-, CN, N3, (R10)2N-C(NR10)-, RlOC(O)-, RlOOC(O)-, -N(RlO) 2 , or Rl lOC(O)NRl -, c) aryl, heterocycle, cycloalkyl, alkenyl, Rl ⁇ O-, Rl lS(0) m -, Rl°C(O)NRl0-, CN, NO2, (Rl°)2N- C(NRl )-, RlOc(O)-, RlO ⁇ C(O)-, N3, -N(RlO)2, or RHOC(O)NRl0-, and d) C
• R6 is independently selected from hydrogen or C1-C6 alkyl
• R8 is independently selected from: a) hydrogen, b) aryl, heterocycle, cycloalkyl, alkenyl, alkynyl, perfluoroalkyl, F, Cl, Br, RlOO-.
• Rl S(0) m -, R10QO)NR 1 0 -, CN. N02, R 10 :N-C(NRl0)-. R ⁇ CfO)-
• Rl oC(O)- N3. -N(R l0)2. or R l lQC(O)NR l0-. 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)-, RlOC(O)-, RlO ⁇ C(O)-, N3, -N(RlO)2, or Rl0 ⁇ C(O)NH-;
• R9 is selected from: a) hydrogen, b) alkenyl, alkynyl, perfluoroalkyl, F, Cl, Br, R IOO-, Rl lS(0) ⁇ r, R1°C(0)NR10-, CN, N02, (Rl°)2N-C-
• RlO is independently selected from hydrogen, C1-C6 alkyl, benzyl and aryl;
• Rl 1 is independently selected from -C6 alkyl and aryl
• Q is a substituted or unsubstituted nitrogen-containing C4-C9 mono or bicyclic ring system, wherein the non-nitrogen containing ring may be a C5-C7 saturated ring;
• V is selected from: a) hydrogen, b) heterocycle. c) arv . 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, 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
• X, Y and Z are independently H2 or O;
• Rla and Rib are independently selected from: a) hydrogen, b) aryl, heterocycle, cycloalkyl, alkenyl, alkynyl, Rl ⁇ O-, Rl lS(0) m -, R!0C(O)NR10-, CN, N ⁇ 2, (Rl°)2N- C(NRlO)-, Rl0c(O)-, RlO ⁇ C(O)-, N3, -N(RlO) 2 , or Rl lOC(O)NRl0-, c) C1-C6 alkyl unsubstituted or substituted by aryl, heterocyclic, cycloalkyl, alkenyl, alkynyl, R ⁇ O-, R ⁇ S(0) ⁇ r, Rl°C(O)NRl -, CN, (R10) 2 N-C(NR10)-, RlOC(O)-, RlO ⁇ C(O)-, N3, -N(Rl )
• R2 and R3 are independently selected from: a) a side chain of a naturally occurring amino acid, b) an oxidized form of a side chain of a naturally occurring amino acid which is: i) methionine sulfoxide, or ii) methionine sulfone, and c) substituted or unsubstituted C1-C2O alkyl, C2-C20 alkenyl, C3-C10 cycloalkyl, aryl or heterocyclic group, wherein the substituent is selected from F, Cl, Br, N(RlO)2, N02, Rl°0-, Rl lS(0) m -, R!0C(O)NR10-,
• R2 and R3 are combined to form - (CH2)s - ;
• R2 or R3 are combined with R6 to form a ring such that
• R4a, R4b, R7a and R7b are independently selected from: a) hydrogen, b) C l -C6 alkyl unsubstituted or substituted by alkenyl, R 1 °0-, R lS(0)m-, Rl°C(O)NRl0., CN, N3, (RlO) 2 N-C(NRlO)-,
• R6 is independently selected from hydrogen or C1-C6 alkyl
• R8 is independently selected from: a) hydrogen, b) aryl, heterocycle, cycloalkyl, alkenyl, alkynyl, perfluoroalkyl, F, Cl, Br, R OO-, Rl lS(0) m -,
• R9 is selected from: a) hydrogen, b) alkenyl, alkynyl, perfluoroalkyl, F. Cl, Br, R 1 OO-, R 1 ] S (O) ⁇ r, R ! 0C(O)NR 10-, CN. N ⁇ 2, (R ⁇ 0 )2N-C- (NRlO)-, R l0C(O)-, RlO ⁇ C(O)-.
• RlO is independently selected from hydrogen, C1-C6 alkyl, benzyl and aryl;
• Rl 1 is independently selected from C1-C6 alkyl and aryl
• Q is a substituted or unsubstituted nitrogen-containing C4-C9 mono or bicychc ring system, wherein the non-nitrogen containing ring may be a C5-C7 saturated ring;
• V is selected from: a) hydrogen, b) heterocycle, c) aryl, d) C1-C20 alkyl wherein from 0 to 4 carbon atoms are replaced with a 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 A is S(0)m;
• Ras famesyl transferase inhibitors are illustrated by the Formula I:
• Rla is independently selected from: hydrogen or Cl-C6 alkyl
• Rib is independently selected from: a) hydrogen, b) aryl, heterocycle, cycloalkyl, Rl°0-, -N(R 1°)2 or alkenyl, c) C 1-C6 alkyl unsubstituted or substituted by aryl, heterocycle, cycloalkyl, alkenyl, Rl ⁇ O-, or -N(Rl°)2;
• R2 and R3 are independently selected from: a) a side chain of a naturally occurring amino acid, b) an oxidized form of a side chain of a naturally occurring amino acid which is: i) methionine sulfoxide, or ii) methionine sulfone.
• Ci-Cio alkyl C2-C10 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)-, RlO ⁇ C(O)-, N3, -N(Rl ) 2 , Rl lOC(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; or
• R2 and R3 are combined to form - (CH2)s - ' ⁇ > or
• R2 or R3 are combined with R6 to form a ring such that
• R4a and R ⁇ a are independently selected from: a) hydrogen, b) C 1 -C6 alkyl unsubstituted or substituted by alkenyl, R IOO-, Rl lS(0)m-, R1°C(0)NR10-, CN, N3, (R10)2N-C(NR10)-,
• R4b and R 7b are hydrogen;
• R5a is selected from: a) a side chain of a naturally occurring amino acid, wherein the amino acid is selected from methionine and glutamine, 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 Cl-CiO alkyl, C2-C10 alkenyl, C3-C10 cycloalkyl, aryl or heterocyclic group, wherein the substituent is selected from F, Cl, Br,
• R5b is selected from: a) hydrogen, and b) C1-C3 alkyl;
• R6 is independently selected from hydrogen or C1-C6 alkyl
• R8 is independently selected from: a) hydrogen, b) C 1 -C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C 1 -C6 perfluoroalkyl, F, Cl, RlOO-, R!0C(O)NR1 -, CN, N02, (RlO) 2 N-C(NRlO)-, RlOc(O)-, RIOOC(O)-, -N(RlO)2, or Rl lOC(O)NRl0-, and c) C1-C6 alkyl substituted by C1-C6 perfluoroalkyl, RlOO-, Rl0C(O)NRl0-, (RlO)2N-C(NRlO)-, RlOc(O)-, RlO ⁇ C(O)-.
• R9 is selected from: a) hydrogen, b) C2-C6 alkenyl, C2-C6 alkynyl, C l -C6 perfluoroalkyl, F, Cl, RlOO-, Rl ls(0) m -, R1°C(0)NR10-, CN, N ⁇ 2, (Rl°)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, R OO-, RHS(0) m -, R1°C(0)NR1 -, CN, (RlO)2N-C(NRl )-, RlOc(O)-, RlOoC(O)-, -N(RlO) 2 , O ⁇ RHOC(O)NR10-;
• RlO is independently selected from hydrogen, C1-C6 alkyl, benzyl and aryl;
• Rl 1 is independently selected from C1-C6 alkyl and aryl
• Q is selected from:
• 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 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 isoquinolinyl;
• X, Y and Z are independently H2 or O;
• Rla is independently selected from: hydrogen or C1-C6 alkyl:
• Rlb is independently selected from: a) hydrogen, b) aryl, heterocycle, cycloalkyl, Rl ⁇ O-, -N(RlO)2 or alkenyl, c) Cl-C6 alkyl unsubstituted or substituted by aryl, heterocycle, cycloalkyl, alkenyl, RlOO-, or -N(RlO)2;
• R2 and R3 are independently selected from: a) a side chain of a naturally occurring amino acid, b) an oxidized form of a side chain of a naturally occurring amino acid which is: i) methionine sulfoxide, or ii) methionine sulfone, c) substituted or unsubstituted C l -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, R10C(0)NR10-, CN, (RlO)2N-C(NRlO)-, RlOc(O)-, RlO ⁇ C(O)-, N3, -N(RlO)2, Rl lOC(O)NRl0- and Cl-Qo alkyl, and d) C1-C6 alkyl substituted with an un
• R2 and R3 are combined to form - (CH2)s - ;
• R2 or R3 are combined with R6 to form a ring such mat
• R4a and R7a are independently selected from: a) hydrogen. b) C l -C6 alkyl unsubstituted or substituted by alkenyl, R 10 ⁇ -, R ⁇ S(0) m -, Rl°C(O)NRl0-, CN, N3, (R1°)2N-C(NR1 )-, RlOC(O)-, RlO ⁇ C(O)-, -N(RlO)2, or RHOC(O)NR10-, c) aryl, heterocycle, cycloalkyl, alkenyl, RlOO-, Rl lS(0)m-, RlOC(0)NRlO-, CN, N ⁇ 2, (Rl°)2N-
• R4b and R ⁇ b are hydrogen
• R5a is selected from: a) a side chain of a naturally occurring amino acid, wherein the amino acid is selected from methionine and glutamine, 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 -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-, (RlO) 2 NC(0)-, CN, (Rl ) 2 N-C(NRl0)-, RlOc(O)-,
• R5b is selected from: a) hydrogen, and b) C1-C3 alkyl;
• R6 is independently selected from hydrogen or C1-C6 alkyl;
• R8 is independently selected from: a) hydrogen, b) C l -C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C l -C6 perfluoroalkyl, F, Cl, RlOO-, R!0C(O)NR10-, CN, N02, (Rl )2N-C(NRl )-, RlOc(O)-, RlO ⁇ C(O)-, -N(RlO)2, or Rl lOC(O)NRl0-, and c) C1-C6 alkyl substituted by C1-C6 perfluoroalkyl, RlOO-,
• R9 is selected from: a) hydrogen, b) C2-C6 alkenyl, C2-C6 alkynyl, C l -C ⁇ perfluoroalkyl, F, Cl, RlOo-, Rl lS(0) ⁇ r, R1°C(0)NR10-, CN, N02, (Rl°)2N- C(NRlO)-, Rl0c(O)-, RlO ⁇ C(O)-, -N(RlO) 2 , or Rl lOC(O)NRl0-, and c) C 1 -C6 alkyl unsubstituted or substituted by C l -C6 perfluoroalkyl, F, Cl, RlOO-, Rl lS(0) m -, R1°C(0)NR1 -, CN, (RlO)2N-C(NRlO)-, RlOC(O)-, RlOOC(O)-, -N(Rl )2,
• 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 a) substituted or unsubstituted C 1 -C8 alkyl or substituted or unsubstituted C5-C8 cycloalkyl, wherein the substituent on the alkyl or cycloalkyl is selected from: 1 ) aryl. 2) heterocycle,
• Rl3 is independently selected from hydrogen and Cl-C6 alkyl
• Rl4 is independently selected from C1-C6 alkyl
• Q is selected from:
• 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) C 1 - 20 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 is S(0)m and V is not hydrogen if . .
• n is a bond
• X, Y and Z are independently H2 or O;
• Rla is independently selected from: hydrogen or Cl-C6 alkyl
• R ib is independently selected from: a) hydrogen. b) arvl. heterocvcle. cvcloalkvl. R l°0-, -N(R l0)2 or alkenyl. c) C1-C6 alkyl unsubstituted or substituted by aryl, heterocycle, cycloalkyl, alkenyl, Rl ⁇ O-, or -N(RlO)2;
• R2 and R3 are independently selected from: a) a side chain of a naturally occurring amino acid, b) an oxidized form of a side chain of a naturally occurring amino acid which is: i) methionine sulfoxide, or ii) methionine sulfone, c) substituted or unsubstituted C 1 -C 10 alkyl, C2-C 10 alkenyl ,
• R2 and R3 are combined to form - (CH2)s - ;
• R2 or R3 are combined with R6 to form a ring such that
• R4a and R ⁇ are independently selected from: a) hydrogen, b) C 1 -C ⁇ alkyl unsubstituted or substituted by alkenyl, R 1 OO-, R l l S(0) m -, Rl0C(O)NR l0-, CN. N3, (R ⁇ 0) 2 N-C(NR10)-, R l0C(O)-, RlOoC(0)-, -N(R lO)2.
• Rl lOC(0)NR lO. c) aryl, heterocycle, cycloalkyl, alkenyl, Rl ⁇ O-, Rl lS(0)m-, R 10 C(O)NRl0-, CN, N ⁇ 2, (R i0 )2N- C(NRlO)-, Rl0c(O)-, Rl ⁇ 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 C3- C 10 cycloalkyl;
• R4b and R?b are hydrogen
• R6 is independently selected from hydrogen or C1-C6 alkyl
• R8 is independently selected from: a) hydrogen, b) C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 perfluoroalkyl, F, Cl, Rl ⁇ O-, Rl n C(O)NRl0-, CN, N ⁇ 2, (RlO)2N-C(NRlO)-, RlOc(O)-, R10OC(O)-, -N(Rl ) 2 , or Rl lOC(O)NRl0-, and c) C 1 -C ⁇ alkyl substituted by C 1 -C6 perfluoroalkyl, R 1 °0-, Rl C(O)NRl0-, (RlO) 2 N-C(NRlO)-, RlOC(O)-,
• R9 is selected from: a) hydrogen, b) C2-C6 alkenyl, C2-C6 alkynyl, C l -C ⁇ perfluoroalkyl, F, Cl,
• R 10 C(O)NRl0-, CN, N02, (Rl°)2N- C(NRl )-, RlOc(O)-, R lO ⁇ C(O)-, -N(Rl0) 2 , or Rl lOC(O)NRl0-, and c) C 1 -C6 alkyl unsubstituted or substituted by C l -C6 perfluoroalkyl, F, Cl, RlOO-, R ] l S(0) m -, R! C(O)NR 10-,
• R lO is independently selected from hydrogen. C j -C ⁇ alkyl. benzyl and aryl;
• Rl 1 is independently selected from -C6 alkyl and aryl
• Q is selected from:
• V is selected from: a) hydrogen, b) heterocycle selected from pyrrolidinyl, imidazolyl, pyridinyl, thiazolyl, pyridonyl, 2-oxopiperidinyl, indolyl, quinohnyl, 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;
• X, Y and Z are independently H2 or O;
• n 0. 1 or 2;
• Rla is independently selected from: hydrogen or Cl-C6 alkyl
• Rib is independently selected from: a) hydrogen, b) aryl, heterocycle, cycloalkyl, R 10 ( 3-, -N(R 10)2 or alkenyl, c) C1-C6 alkyl unsubstituted or substituted by aryl, heterocycle, cycloalkyl, alkenyl, R °0-, or -N(Rl°)2;
• R2 and R3 are independently selected from: a) a side chain of a naturally occurring amino acid, b) an oxidized form of a side chain of a naturally occurring amino acid which is: i) methionine sulfoxide, or ii) methionine sulfone, c) substituted or unsubstituted Cl-ClO alkyl, C2-C10 alkenyl, C3-C10 cycloalkyl, aryl or heterocyclic group, wherein the substituent is selected from F, Cl, Br,
• R2 and R3 are combined to form - (CH2)s - ;
• R2 or R3 are combined with R6 to form a ring such that
• R4a and R ⁇ a are independently selected from: a) hydrogen, b) C 1 -C6 alkyl unsubstituted or substituted by alkenyl, R 1 O-, R ⁇ S(0) m -, Rl0C(O)NRl0-, CN, N3, (R10) 2 N-C(NR10)-, Rl C(O)-, RI OC(O)-, -N(Rl0)2, or Rl l ⁇ C(O)NRl0-, c) aryl, heterocycle, cycloalkyl, alkenyl, Rl O-, Rl l S(0)m-, R 10 C(O)NRl0-, CN. N02, (Rl ) 2 N-
• R6 is independently selected from hydrogen or C1-C6 alkyl
• R8 is independently selected from: a) hydrogen, b) C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 perfluoroalkyl, F, Cl, Rl O-, R!0C(O)NR1 -, CN, NO2, (Rl )2N-C(NRl )-, RlOc(O)-, Rl oC(O)-, -N(RlO) 2 , or RH ⁇ C(O)NRl0-, and c) C1-C6 alkyl substituted by C1-C6 perfluoroalkyl, Rl°0-, Rl0C(O)NRl0-, (RlO) 2 N-C(NRl )-, Rl0c(O)-, Rl ⁇ C(O)-, -N(RlO) 2 , or Rl l ⁇ C(O)NRl0- ;
• R9 is selected from: a) hydrogen, b) C2-C6 alkenyl, C2-C6 alkynyl, C 1 -C ⁇ perfluoroalkyl, F, Cl, RlOO-, Rl lS(0)m-, R1°C(0)NR10-, CN, N ⁇ 2, (Rl°)2N- C(NRl )-, RlOC(O)-, RIOOC(O)-, -N(Rl ) 2 , or Rl lOC(O)NRl0-, and c ) C l -C ⁇ alkyl unsubstituted or substituted by C l -C ⁇ perfluoroalkyl, F, Cl, Rl O-, Rl lS(0) m -, R 10 C(O)NRl0-, CN, (RlO) 2 N-C(NRlO)-, RlOC(O)-, RlOoC(O)-, -N(Rl ) 2 ,
• RlO is independently selected from hydrogen, C1-C6 alkyl, benzyl and aryl;
• Rl 1 is independently selected from C1 -C6 alkyl and aryl
• Q is selected from:
• V is selected from: a) hydrogen, b) heterocycle selected from py ⁇ olidinyl, imidazolyl, pyridinyl, thiazolyl, pyridonyl, 2-oxopiperidinyl, indolyl, quinolinyl, isoquinolinyl, and thienyl, c) aryl, d) Ci-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 A2 is S(0)m",
• W is a heterocycle selected from py ⁇ olidinyl, imidazolyl, pyridinyl, thiazolyl, pyridonyl, 2-oxopiperidinyl, indolyl, quinolinyl, or isoquinolinyl;
• X, Y and Z are independently H2 or O;
• the prefe ⁇ ed compounds of this invention are as follows:
• amino acids which are disclosed are identified both by conventional 3 letter and single letter abbreviations as indicated below:
• the compounds of the present invention may have asymmetric centers and occur as racemates, racemic mixtures, and as individual diastereomers, with all possible isomers, including optical isomers, being included in the present invention.
• 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, benzofiiryl, benzothiazolyl, benzothienyl, benzoxazolyl, chromanyl, cinnolinyl, dihy drobenzofuryl, dihydro- benzothienyl, dihydrobenzothiopyranyl, dihydrobenzothio-pyranyl sulfone, furyl, imidazolidinyl, 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(C_ - C6 alkyl)2, N ⁇ 2, CN, (Cl-C6 alkyl.
• 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.
• a cyclic amine moiety include, but are not limited to, the following specific structures:
• substitution on the cyclic amine moiety by R ⁇ a and R ⁇ b may be on different carbon atoms or on the same carbon atom.
• R3 and R4 are combined to form - (CH2)s - ⁇ cyclic moieties are formed.
• Examples of such cyclic moieties include, but are not limited to:
• cyclic moieties as described hereinabove for R and R ⁇ are formed.
• cyclic moieties may optionally include a heteroatom(s). Examples of such heteroatom-containing cyclic moieties include, but are not limited to:
• the phrase "nitrogen containing C4-C9 mono or bicychc ring system wherein the non-nitrogen containing ring may be a C5-C7 saturated ring" which defines moiety "Q" of the instant invention includes but is not limited to the following ring systems:
• 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(Rl )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.
• Rla and Rib are independently selected from: hydrogen, -N(R8)2, R 8 C(0)NR8- or -C6 alkyl unsubstituted or substituted by -N(R8)2, R80- or R8C(0)NR8-.
• R2 is the sidechain of glycine (hydrogen).
• R3 is selected from: a) a side chain of a naturally occurring amino acid, b) substituted or unsubstituted C1-C2O alkyl, wherein the substituent is selected from F, Cl, Br, N(RlO)2, N ⁇ 2, Rl°0-, Rl lS(0) ⁇ r.
• R 10 C(O)NRl0_ is independently selected from: hydrogen, -N(R8)2, R 8 C(0)NR8- or -C6 alkyl unsubstituted or substituted by -N(R8)2, R80- or R8C(0)NR8-.
• R2 is the sidechain of glycine (hydrogen).
• R3
• R3 is combined with R6 to form py ⁇ olidinyl ring.
• R4a, R4b, R7a and R 7b are independently selected from: hydrogen, C 1-C6 alkyl. aryl and benzyl.
• R and R ⁇ b are independently selected from: a side chain of a naturally occurring amino acid, methionine sulfoxide, methionine sulfone and unsubstituted or substituted C1-C6 alkyl .
• R6 is: hydrogen or is combined with R3 to form py ⁇ olidinyl ring.
• R8 is selected from: hydrogen, perfluoroalkyl, F,
• R ⁇ is hydrogen
• Rl is selected from H, C1-C6 alkyl and benzyl.
• R 1 is selected from C 1 -C6 alkyl and benzyl.
• Al and A2 are independently selected from: a bond, -C(O)NRl0-, -NR10C(O)-, O, -N(R10)-, -S(O)2N(Rl0)- and-
• Q is a py ⁇ olidinyl ring.
• V is selected from hydrogen, heterocycle and aryl.
• n, p and r are independently 0, 1, or 2.
• t is 3.
• the pharmaceutically acceptable salts of the compounds of this invention can be synthesized from the compounds of this invention which contain a basic moiety conventional chemical methods. Generally, the salts are prepared by ion exchange chromatography or by reacting the free base with stoichiometric amounts or with an excess of the desired 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 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 E Preparation of a reduced subunit by borane reduction of the amide moiety.
• Reaction Schemes A-E illustrate bond-forming and peptide modifying reactions inco ⁇ orating acyclic peptide units. It is well understood that such reactions are equally useful when the - NHC(RA) - moiety of the reagents and compounds illustrated is replaced with the following moiety:
• Reaction B Preparation of reduced peptide subunits by reductive alkylation
• RA is R2, R3, R5a or R5b a s previously defined; R4a and R4b are as previously defined; and R is an appropriate protecting group for the carboxylic acid.
• Reaction Schemes F - M illustrate reactions wherein the non- sulfhydryl-containing moiety at the N-terminus of the compounds of the instant invention is attached to an acyclic peptide unit which may be further elaborated to provide the instant compounds. It is well understood that such reactions are equally useful when the - NHC(R A ) - moiety of the reagents and compounds illustrated is replaced with the following moiety:
• Schemes A and C can be reductively alkylated with a variety of aldehydes, such as I, as shown in Reaction Scheme F.
• the aldehydes can be prepared by standard procedures, such as that described by O. P. Goel, U. Krolls, M. Stier and S. Kesten in Organic Syntheses. 1988, 67, 69-75, from the appropriate amino acid (Reaction Scheme F).
• the reductive alkylation can be accomplished at pH 5-7 with a variety of reducing 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 HI is isolated in the salt form, for example, as a trifluoroacetate, hydrochloride or acetate salt, among others.
• the product diamine ffl can further be selectively protected to obtain IV, which can subsequently be reductively alkylated with a second aldehyde to obtain V. Removal of the protecting group, and conversion to cyclized products such as the dihydroimidazole VII can be accomplished by literature procedures.
• the protected dipeptidyl analog intermediate can be reductively alkylated with other aldehydes such as l-trityl-4- carboxaldehyde or l-trityl-4-imidazolylacetaldehyde, to give products such as VIH (Reaction Scheme G).
• T e trityl protecting group can be removed from Vffl to give IX, or alternatively, VTII can first be treated with an alkyl halide then subsequently deprotected to give the alkylated imidazole X.
• the dipeptidyl analog intermediate can be acylated or sulfonylated by standard techniques. The imidazole acetic acid XI can be converted to the acetate
• XIII by standard procedures, and XIII can be first reacted with an alkyl halide. then treated with refluxing methanol to provide the regiospecifically alkylated imidazole acetic acid ester XIV.
• Hydrolysis and reaction with the protected dipeptidyl analog intermediate in the presence of condensing reagents such as l-(3-dimethylaminopropyl)-3- ethylcarbodiimide (EDC) leads to acylated products such as XV.
• condensing reagents such as l-(3-dimethylaminopropyl)-3- ethylcarbodiimide (EDC) leads to acylated products such as XV.
• the protected dipeptidyl analog intermediate is reductively alkylated with an aldehyde which also has a protected hydroxyl group, such as XVI in Reaction Scheme I
• the protecting groups can be subsequendy removed to unmask the hydroxyl group (Reaction Schemes I, J).
• the alcohol can be oxidized under standard conditions to e.g. an aldehyde, which can then be reacted with a variety of organometallic reagents such as Grignard reagents, to obtain secondary alcohols such as XX.
• 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 K), or tertiary amines.
• the Boc protected amino alcohol XVm can also be utilized to synthesize 2-aziridinylmethylpiperazines such as XXIII (Reaction Scheme L). Treating XV ⁇ i with l, -sulfonyldiimidazole and sodium hydride in a solvent such as dimethylformamide 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 xxrv .
• a nucleophile such as a thiol
• the protected dipeptidyl analog intermediate 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 M.
• R' 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 XXX ⁇ produced.
• Reaction Schemes F- M may be employed using other peptidyl analog intermediates such as those whose synthesis is illustrated in Reaction Schemes B - E.
• Reaction Schemes N-R illustrate syntheses of suitably substituted aldehydes useful in the syntheses of the instant compounds wherein the variable W is present as a pyridyl moiety. Similar synthetic strategies for preparing alkanols that inco ⁇ orate other heterocyclic moieties for variable W are also well known in the art.
• the instant 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, myeloid leukemias and neurological tumors. Such tumors may arise by mutations in the ras genes themselves, mutations in the proteins that can regulate Ras formation (i.e., neurofibromin (NF-1), neu, scr, abl, lck, fyn) or by other mechanisms.
• the compounds of the instant invention inhibit famesyl- protein transferase and the famesylation of the oncogene protein Ras.
• the instant compounds may also inhibit tumor angiogenesis, thereby affecting the growth of tumors (J. Rak et al. Cancer Research, 55:4575- 4580 (1995)). Such anti-angiogenesis properties of the instant compounds may also be useful in the treatment of certain forms of blindness related to retinal vascularization.
• the compounds of this invention are also useful for inhibiting other proliferative diseases, both benign and malignant, wherein Ras proteins are aberrantly activated as a result of oncogenic mutation in other genes (i.e., the Ras gene itself is not activated by mutation to an oncogenic form) with said inhibition being accomplished by the administration of an effective amount of the compounds of the invention to a mammal in need of such treatment.
• a component of NF-1 is a benign proliferative disorder.
• the instant compounds may also be useful in the treatment of certain viral infections, in particular in the treatment of hepatitis delta and related viruses (J.S. Glenn et al. Science, 256: 1331-1333 (1992).
• the compounds of the instant invention are also useful in the prevention of restenosis after percutaneous transluminal coronary angioplasty by inhibiting neointimal formation (C. Indolfi et al. Nature medicine, 1 :541-545( 1995).
• the instant compounds may also be useful in the treatment and prevention of polycystic kidney disease (D.L. Schaffner et al. American Journal of Pathology, 142:1051-1060 (1993) and B. Cowley, Jr. et z ⁇ .FASEB Journal, 2:A3160 (1988)).
• 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 administration 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
• 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.
• FPTase for example a tetrapeptide having a cysteine at the amine terminus
• famesyl pyrophosphate for example a tetrapeptide having a cysteine at the amine terminus
• the chemical content of the assay mixtures may be determined by well known immunological, radiochemical or chromatographic techniques.
• 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 sufficiendy potent inhibitor i.e., one that has a Ki substantially smaller than the concentration of enzyme in the assay vessel
• concentration of a sufficiendy potent inhibitor 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.
• 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.
• Step A Preparation of Diethyl l-acetyl-5-hydroxy-3- ethvlpvrrolidine-2.2-dicarboxvlate
• Step B Preparation of Diethyl l-acetyl-3-ethylpyrrolidine-2,2- dicarboxylate
• Step D Preparation of N-[(tert-Buty ⁇ oxy)ca ⁇ bony ⁇ ]-cis:trans-3- ethvlproline methvl ester
• 3-Ethylproline hydrochloride (Cis:Trans Mixture) (20 g, 0.11 mol) was dissolved in CH3OH (200 mL), and the solution was saturated with HCI gas, then stirred at 23 °C for 24 h. Argon was bubbled through the solution to remove excess HCI. The solution was treated with NaHC03 (>84 g) to a pH of 8, then di- rf-butyl dicarbonate (25.1 g, 0.115 mol) dissolved in CH3OH (20 mL) was added slowly. After stirring for 18 h at 23°C, the mixture was filtered, the filtrate concentrated, and the residue triturated with EtOAc, filtered again, and concentrated to give the title compound as an oil.
• Step E Preparation of N-[(r_ , rt-Butyloxy)carbonyl]-rr__j.5-3- ethylproline and N-[(t -Butyloxy)carbonyl]-c/s-3- ethylproline methyl ester
• N-[(rt?rr-Butyloxy)carbonyl]- ⁇ .tr_2/.5-3-ethylproline methyl ester (29.1 g, 0.1 13 mol) was dissolved in CH3OH ( 1 14 mL) with cooling to 0°C. then treated with 1 N NaOH ( 1 14 mL). After stirring for 20 h at 23°C, the solution was concentrated to remove the CH3OH and then extracted with EtOAc (3 x). The organic layers were combined, dried (MgS ⁇ 4), filtered, and concentrated to give 12.8 g of N-[(tert- Butyloxy)carbonyl]-ris-3-ethylproline methyl ester as an oil.
• Step F Preparation of 3(S)-Ethyl-2(S .-proline hydrochloride
• N-[(te ⁇ Butyloxy)carbonyl]-fr ⁇ ns-3-ethylproline (15.5 g, 0.064 mol), S- ⁇ -methylbenzylamine (9.03 mL, 0.070 mol), HOBT (10.73 g, 0.70 mol), and N-methylmorpholine (8 mL, 0.076 mol) were dissolved in CH2CI2 (150 mL) with sit ⁇ ing in an ice-H2 ⁇ bath, treated with EDC (13.4 g, 0.070 mol) stirred at 23°C for 48 h.
• N-[( /?-Butyloxy)carbonyl- 3(S)-ethyl-2(S)-proline was dissolved in EtOAc (50 mL) and the solution was saturated with HCI gas with cooling in an ice-H2 ⁇ bath. The solution was stoppered and stirred at 0°C. for 3 hr. Argon was bubbled through the solution to remove excess HCI, and the solution was concentrated to dryness to give 3(S)-ethyl-2(S)-proline hydrochloride.
• Step Q N-r(fgrf-Butvloxv.carbonvn-3(S .-ethvl-2(S .-prolinol 3(S)-Ethyl-2(S)-proline hydrochloride containing ⁇ - memylbenzyl ⁇ imine (2.0 g, 0.0128 mol) was dissolved in dioxane (10 mL) and H2 ⁇ (10 mL) with stirring and cooling to 0°C. N,N- diisopropylethylamine (2.2 mL, 0.0128 mol) and di-terf-butyl- dicarbonate (2.79 g, 0.0128 mol) were added and stirring was continued at 23°C for 48 h.
• N-[(tert-Butyloxy)carbonyl]-3(S)-ethyl-2(S)-proline (1.6 g, 6.58 mmol) was dissolved in dry THF (10 mL) and treated with borane (IM in THF, 12.5 mL, 12.5 mmol) with stirring at 0 °C for 2 h, then 23°C for 1 h.
• the solution was cooled to 0°C, treated with H2O (20 mL), and extracted with EtOAc (2 x 30 mL). The organics were washed with brine, satd NaHC ⁇ 3, H2O, dried (MgS ⁇ 4). filtered and concentrated to give a viscous oil. The oil was dissolved in CH2CI .
• Step H N-r(r -Butyloxy.carbonyll-3(S .-ethyl-2(S)-prorinal
• N-[(f ⁇ rr-Butyloxy)carbonyl-3(S)-ethyl-2(S)-prolinol (0.638 g, 2.78 mmol) and E_3N (1.4 mL, 9.74 mmol) were dissolved in dry CH2CI2 (10 mL) with stirring and cooling to -10°C and treated dropwise with a solution of S ⁇ 3.pyr (1.33 g, 8.35 mmol) in dry DMSO (5 mL) keeping the reaction mixture temperature at ⁇ 0°C. The mixture was stirred at 0°C. for 20 min then at 5°C for 20 min, and at 15°C for 1 h, then poured into ice-cold 0.5 N HCI and the layers separated.
• Step I N-[( rt-Butyloxy)carbonyl-3(S)-ethylpy ⁇ o_idin-2(S)- ylmethyl 1-proline methyl ester
• N-[(rerf-Butyloxy)carbonyl]-3(S)-ethyl-2(S)-prolinal (0.315 g, 0.0014 mol) and proline methyl ester hydrochloride (0.233 g, 0.0014 mol) were dissolved in MeOH (5 mL) at ambient temperature under argon with cooling in an ice-H2 ⁇ bath, and treated with sodium cyanoborohydride (0.131 g, 0.002 mol) with stirring.
• Step J N-[(fcrt-Butyloxy)carbonyl-3(S)-ethylpyrrohdin-2(S)- ylmethyll-proline
• N-[(rf?rf-Butyloxy)carbonyl-3(S)-ethylpyrrohdin-2(S)- ylmethylj-proline methyl ester 0.081 g, 0.238 mmol) was dissolved in CH3OH (2 mL), cooled to 0°C and treated with IN NaOH solution (0.952 mL, 0.952 mmol). After stirring at 23°C for 3h, the solution was neutralized with IN HCI (0.952 mL, 0.952 mmol), concentrated to remove the CH3OH, then lyophilized and the residue used as is.
• Step K N-[(terf-Butyloxy)carbonyl-3(S)-ethylpyrtolidin-2(S)- ylmethyl ⁇ -prolyl-methionine methyl ester
• Step L N-[3(S)-Ethylpyrrolidin-2(S)-ylmethyl]-prolyl-methionine methyl ester hydrochloride
• HCI gas was bubbled into a solution of N-[(tert- butyloxy)carbonyl-3(S)-ethylpyrrolidin-2(S)-ylmethyl]-prolyl methioninine methyl ester (0.085 g, 0.18 mmol) in EtOAc (5 mL) with stirring and cooling in an ice-H2 ⁇ bath until saturation.
• EtOAc 5 mL
• the solution was stoppered and stirred at 0°C for 2 h, then purged with Ar and concentrated to give the title compound as a yellow foam.
• Step M N-[ l-( lH-Imidazol-4-ylacetyl)-3(S)-ethylpyrrolidin-2(S)- lmethyll-prolyl-methionine methyl ester N-[3(S)-Ethylpyrrolidin-2(S)-ylmethyl]-prolyl methionine methyl ester hydrochloride (0.075 g, 0.169 mmol), lH-imidazol-4- ylacetic acid (0.052 g, 0.253 mmol), HOBT (0.34 g, 0.253 mmol), EDC (0.49 g, 0.253 mmol) and Et3N ( 0.176 mL, 1.27 mmol) were dissolved in DMF (4 mL) and stirred at 23°C for 18 h.
• Step N N-[l-(lH-Imidazol-4-ylacetyl)-3(S)-ethylpy_ ⁇ ohdin-
• N-[l-(lH-Imidazol-4-ylacetyl)-3(S)-ethylpyrrolidin-2(S)- ylmethyl]-prolyl-methionine methyl ester (0.020 g, 0.042 mmol) was dissolved in CH3OH (2 mL) at 0°C and treated with IN NaOH solution (0.167 mL, 0.167 mmol) with stirring. After 5 h at 23°C IN HCI (0.167 mL, 0.167 mmol) was added and the mixture was purified by preparative RP HPLC on a Vydac column eluting with 0.1% TFA/CH3CN: 0.1% TFA/ H2 ⁇ gradient to give the title compound.
• Step A N-[(t-Butyloxycarbonyl)-pyrrolidin-2(S)-ylmethyl]-3(S)- ethvl-proline
• 3(S)-Ethyl-2(S)-proline hydrochloride (from Example 1, Step F) (2.33 g, 0.013 mol) was dissolved in CH3OH (20 mL), treated with 3 A molecular sieves (2 g) and KOAc (1.27 g, 0.013 mol) to adjust the pH of the reaction mixture to 4.5-5, then N-[(tert- Butyloxy)carbonyl-prolinal (Pettit et al., J. Org. Chem. (1994) 59, [21] 6287-95) (3.36 g, 0.017 mol) was added, and the mixture was stirred for 16 hrs at room temperature.
• reaction mixture was filtered, quenched with aq satd NaHC ⁇ 3 (5 mL) and concentrated to dryness. The residue was extracted with CHCI3. The extract was dried (MgS ⁇ 4), filtered, and concentrated to give the title compound and inorganic salts.
• Step B N-[(t-Butyloxycarbonyl)-pyrrolidin-2(S)-ylmethyl]-3(S)- ethyl-prolyl-methionine isopropyl ester
• N-[(t-Butyloxycarbonyl)-pyrrohdin-2(S)-ylmethyl]-3(S)- ethyl-prolyl-methionine isopropyl ester (1.38 g, 0.0028 mol) was dissolved in EtOAc (40 mL), cooled to -20°C, saturated with HCI gas, and stirred at 0°C. for 1.25 hr, and room temperature for 0.25 hr.
• Step D lH-Imidazole-4- acetic acid methyl ester hydrochloride A solution of lH-imidazole-4-acetic acid hydrochloride
• Step E l-(Triphenylmethyl)-lH-imidazol-4-ylacetic acid methyl ester
• DMF dimethyl formamide
• triethylamine 57.2 ml, 0.412mol
• tribenzyl bromide 55.3g, 0.171mol
• the reaction mixture was diluted with ethyl acetate (EtOAc) ( 1 1) and water (350 ml). The organic phase was washed with sat. aq.
• Step F [ 1 -(4-Cyanobenzyl)- 1 H-imidazol-5-yl]acetic acid methyl ester
• l-(Triphenylmethyl)-lH-imidazol-4- ylacetic acid methyl ester 8.00g, 20.9mmol
• bromo-p-toluonitrile 4.10g, 20.92 mmol
• the reaction was cooled to room temperature and the resulting imidazolium salt (white precipitate) was collected by filtration. The filtrate was heated at 55 °C for 18hr.
• Step G ⁇ 1 -(4-CvanobenzylV 1 H-imidazol-5-vnacetic acid
• Step H N-[l-[l -(4-Cyanobenzyl)- lH-imidazol-5-ylacetyl]- pyr ⁇ olidin-2(S)-ylmethyl]-3(S)-ethyl-prolyl-methionine isopropyl ester
• Step A N-[l-[l -(4-Cyanobenzyl)- lH-imidazol-5-ylacetyl]- pyrtolicUn-2(S)-ylmethyl]-3(S)-ethyl-prolyl-methionine methyl ester
• Step B N-[ 1 -[ 1 -(4-Cyanobe ⁇ zyl)- lH-_midazol-5-l)acetyl]pyI ⁇ olidin- 2 S ⁇ -ylmethyl ⁇ -3 S.-e-hyl-prolyl-methio- ⁇ ne trifluoroacetate
• Step A N-[2(S)-( 1 H-Imidazol-4-ylacetyl-amino)-3(S)- ethylpentyll-prolyl-methionine methyl ester
• N-(t-Butyloxycarbonyl)-isoleucinal N-[(t- Butyloxy)carbonyl]-3(S)-ethyl-2(S)-prolinal in Step I
• the title compound was prepared.
• Step B N-[2(S)-(lH-Imidazol-4-ylacetyl-amino)-3(S)- methylpentvll-prolvl-methionine
• Step A N-[ 1 -( 1 H-Imidazol-4-ylacetyl)-py_ ⁇ olidin-2(S)-ylmethyl]- prolyl-methionine methyl ester
• Step B N-[ 1 -( lH-Imidazol-4-ylacetyl)-pyrrohdin-2(S)-ylmethyl]- prolyl-methionine
• Step A 1 -(4-Nitrobenzyl)- 1 H-imidazol-4-ylacetic acid methyl ester and l-(4-Nitrobenzvl)-lH-imidazol-5-ylacetic acid methyl ester (3:1 mixture.
• sodium hydride 50% in mineral oil, 99 mg
• Step B 1 -(4-Nitrobenzyl)- l H-imidazol-4-ylacetic acid hydrochloride and 1 -(4-Nitrobenzyl)- l H-imidazol-5-ylacetic acid (3: 1 mixture)
• a solution of a mixture of 1 -(4-Nitrobenzyl)- 1H- imidazol-4-ylacetic acid methyl ester and 1 -(4-Nitrobenzyl)- 1H- imidazol-5-ylacetic acid methyl ester 3:1 mixture, 216 mg, 0.785 mmol) in methanol (3 ml) and tetrahydrofuran (3 ml) under argon was added 1.0 M sodium hydroxide ( 1.18 ml, 1.18 mmol) and stirred for 18 h.
• Step C N-[ 1 -( 1 -(4-Nitrobenzyl)- 1 H-imidazol-4-ylacetyl]pyrrolidin- 2(S)-ylmethyl]-3(S)-ethyl-prolyl-methionine methyl ester bis trifluoroacetate and N-[l-(l -(4-Nitrobenzyl)- lH-imidazol-5- ylacetyl]pyrrolidin-2(S)-ylmethyl]-3(S)-ethyl-prolyl methionine methvl ester bis trifluoroacetate
• Step D N- [ 1 -( 1 -(4-Nitrobenzyl)- 1 H-imidazol-4-ylacetyl]pyrroUdin- 2(S)-ylmethyl]-3(S)-ethyl-prolyl-methionine bis ttifluQroacet ⁇ te
• N- [ 1 -( 1 -(4-nitrobenzy 1)- 1 H-imidazol-4- ylacetyl]pyrroUdin-2(S)-ylmethyl]-3(S)-ethyl-prolyl methionine methyl ester bis trifluoroacetate (0.023 mmol) in methanol (1 ml ) at room temperature is added l.ON lithium hydroxide (135 ⁇ l, 0.135 mmol). This solution is stirred for 4 h and treated with trifluoroacetic acid (100 ⁇ l). This mixture is purified by preparative HPLC using chromatography method A to give the title
• Step E N- [ 1 -( 1 -(4-Nitrobenzyl)- 1 H-imidazol-5-ylace tyljpyrrolidin- 2(S)-ylmethyl]-3(S)-ethyl-prolyl-methionine bis trifluoroacetate
• Step A l -( l-Farnesyl .-l H-imidazol-5-ylacetic acid methyl ester
• Step B N-[ 1 -( 1 -( 1 -Famesyl)- 1 H-imidazol-5-ylacetyl)pyrrolidin- 2(S)-ylmethyl]-3(S)-ethyl-prolyl methionine methyl ester bis trifluoroacetate
• Step C N-[ 1 -( 1 -( 1 -Famesyl)- 1 H-imidazol-5-ylacetyl)pyrrolidin- 2(S)-ylmethyl]-3(S)-ethyl-prolyl methionine bis trifluoroacetate
• Step N but using the methyl ester prepared as described in Step B provides the title compound.
• Step A N-[ 1 -( 1 -( 1 -Geranyl )- 1 H-imidazol-5-ylacetyl)pyrrolidin-
• Step B N-[ 1-(1-(1 -Geranyl)- lH-imidazol-5- ylacetyl)py olidin-2(S)-ylmethyl]-3(S)-ethyl-prolyl- methionine bis trifluoroacetate
• Step A N-[ 1 -( 1 -(4-Methoxybenzyl)- 1 H-imidazol-5- yl)acetyl)pyrrolidin-2(S)-ylmethyl]-3(S)-ethyl-prolyl- methionine methyl ester bis trifluoroacetate
• Step B N-[l-(l -(4-Methoxybenzyl)- lH-imidazol-5- ylacetyl)py olidin-2(S)-ylmethyl]-3(S)-ethyl-prolyl- methionine bis trifluoroacetate
• Step A N-[l-(l-(2-Naphthylmethyl)-lH-imidazol-5-ylacetyl]3(S)- ethylpy_ ⁇ ohdin-2(S)-ylmethyl]-prolyl-methionine methyl ester bis trifluoroacetate
• Steps B-D but using 2-(bromomethyl)naphthylene in place of 4-nitrobenzylbromide provided the title compound.
• Step B N-[l-(l-(2-Naphthylmethyl)-lH-imidazol-5- ylacetyl]pyrt ⁇ lidin-2(S)-ylmemyl]-3(S)-ethyl-prolyl- methionine bis trifluoroacetate
• Step B Methyl 2(S)-benzyloxycarbonylamino-3-acetylamino- propionate
• pyridine 20 mL
• acetic anhydride 5 mL
• the residue was partitioned between ethyl acetate and water.
• the ethyl acetate layer was extracted w/ 50 mL each of 2% potassium hydrogen sulfate, saturated sodium bicarbonate, saturated sodium chloride, dried over magnesium sulfate and concentrated in vacuo.
• Step D N-[ 1 -( 1 H-Imidazol-4-ylacetyl)-pyrrolidin-2(S)-ylmethyl]- 3(S)-ethyl-prolyl-( ⁇ -acetylamino)alanine methyl ester trifluoroacetate Following the procedures outlined in Examples 1 and 2, but substituting the methyl 2(S)-amino-3-acetylaminopropionate of Step C for methionine methyl ester hydrochloride in Example 1, Step K, the title compound is prepared.
• Step E N-[ l -( lH-Imidazol-4-ylacetyl)-pyrrolidin-2(S)-ylmethyl]-
• Bovine FPTase was assayed in a volume of 100 ⁇ l containing 100 mM _V- (2-hydroxy ethyl) piperazine-_V'-(2-ethane sulfonic acid) (HEPES), pH 7.4, 5 m MgCl2, 5 mM dithiothreitol (DTT), 100 mM [3H]-farnesyl diphosphate ([ 3 H]-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 harvester, 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.
• TCA trichloroacetic acid
• 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 51:112-111, (1991). Cells in 10 cm dishes at 50-75% confluency are treated with the test compound (final concentration of solvent, methanol or dimethyl sulfoxide, is 0.1%). After 4 hours at 37°C, the cells are labelled in 3 ml methionine-free DMEM supple-meted with 10% regular DMEM, 2% fetal bovine serum and 400 mCi[35S]methionine (1000 Ci/mmol).
• 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 antipain/0.5 mM PMSF) and the lysates cleared by centrifugation at 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 antipain/0.5 mM PMSF) and the lysates cleared by centrifugation at 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 antipain/0.5 mM PMSF) and the lys
• 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 famesylated 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 serum) over a bottom agarose layer (0.6%). Both layers contain 0.1% methanol or an appropriate concentration of the instant compound (dissolved in methanol at 1000 times the final concentration used in the assay). The cells are fed twice weekly with 0.5 ml of medium A contaimng 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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