EP0891349A1 - Inhibiteurs de farnesyl-proteine transferase - Google Patents

Inhibiteurs de farnesyl-proteine transferase

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Publication number
EP0891349A1
EP0891349A1 EP97917599A EP97917599A EP0891349A1 EP 0891349 A1 EP0891349 A1 EP 0891349A1 EP 97917599 A EP97917599 A EP 97917599A EP 97917599 A EP97917599 A EP 97917599A EP 0891349 A1 EP0891349 A1 EP 0891349A1
Authority
EP
European Patent Office
Prior art keywords
substituted
alkyl
aryl
cycloalkyl
unsubstituted
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP97917599A
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German (de)
English (en)
Other versions
EP0891349A4 (fr
Inventor
Christopher J. Dinsmore
Theresa M. Williams
Jeffrey Bergman
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Merck and Co Inc
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Merck and Co Inc
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Publication date
Priority claimed from GBGB9613461.4A external-priority patent/GB9613461D0/en
Application filed by Merck and Co Inc filed Critical Merck and Co Inc
Publication of EP0891349A1 publication Critical patent/EP0891349A1/fr
Publication of EP0891349A4 publication Critical patent/EP0891349A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms

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 ⁇ j, Ki4b-ras and N-ras) are found in many human cancers, including colorectal carcinoma, exocrine pancreatic carcinoma, and myeloid leukemias. The protein products of these genes are defective in their GTPase activity and constitutively transmit a growth stimulatory signal.
  • Ras must be localized to the plasma membrane for both normal and oncogenic functions. At least 3 post-translational modifications are involved with Ras membrane localization, and all 3 modifications occur at the C-terminus of 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 570:583-586 (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.
  • 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. James, et al., have also suggested that there are farnesylated proteins of unknown structure and function in addition to those listed above.
  • Famesyl-protein transferase 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: 1 133- 1 139 (1990); Manne et al, Proc. Natl. Acad. Sci USA, 87:1541-1545 (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
  • 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 altemate 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 peptidomimetic 2,5- diketopiperazine-containing compounds which inhibit the famesyl- protein transferase.
  • the instant compounds lack a thiol moiety and thus offer 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 the oncogene protein Ras.
  • the inhibitors of famesyl-protein transferase are illustrated by the formula A:
  • Rl a and Rib are independently selected from: a) hydrogen, b) aryl, heterocycle, C3-C10 cycloalkyl, C2-C6 alkenyl, C2- C6 alkynyl, RlOO-, Rl lS(0) m -, R 1°C(0)NR10-, (RlO) 2 NC(0)-, Rl0 2 N-C(NRl )-, CN, NO2, Rl°C(0)-, N3, -N(RlO)2, or R110C(0)NR 10-, c) unsubstituted or substituted C1-C6 alkyl wherein the substitutent on the substituted C1 -C6 alkyl is selected from unsubstituted or substituted aryl, heterocyclic, C3-C10 cycloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, RlOO-, Rl lS(0) m -, R 1°C(0)NR10-, (
  • R2 and R4 are independently selected from: H; unsubstituted or substituted Cl-8 alkyl, unsubstituted or substituted C2-8 alkenyl, unsubstituted or substituted C2-8 alkynyl, unsubstituted or substituted aryl,
  • halogen e) CN, f) aryl or heteroaryl, g) perfluoro-Cl -4 alkyl, or h) SR 6a , S(0)R6a, S ⁇ 2R 6a ,
  • R3 and R5 are selected from H and CH3; or
  • R2 and R ⁇ or R ⁇ and R ⁇ attached to the same C atom are combined to form - (CH2)u - wherein one of the carbon atoms is optionally replaced by a moiety selected from: O, S(0) m , -NC(O)-, and -N(CORlO)- ;
  • R6, R7 and R7a are independently selected from: H; Cj-4 alkyl, C3-6 cycloalkyl, heterocycle, aryl, aroyl, heteroaroyl, arylsulfonyl, heteroarylsulfonyl, unsubstituted or substituted with: a) Cl -4 alkoxy, b) aryl or heterocycle, c) halogen, d) HO,
  • R6 and R may be joined in a ring;
  • R7 and R7a may be joined in a ring;
  • R6 i s selected from: Cl -4 alkyl, C3-6 cycloalkyl, heterocycle, aryl, unsubstituted or substituted with: a) Cl -4 alkoxy, b) aryl or heterocycle, c) halogen, d) HO, pH
  • R8 is independently selected from: a) hydrogen, b) aryl, heterocycle, C3-C10 cycloalkyl, C2-C6 alkenyl, C2- C6 alkynyl, perfluoroalkyl, F, Cl, Br, Rl OO-, Ri l S(0) m -, Rl0C(O)NR l0-, (RlO) 2 NC(0)-, Rl0 N-C(NRlO)-, CN, N02, Rl°C(0)-, N3, -N(RlO)2, or Rl 10C(0)NR 10-, and c) C1-C6 alkyl unsubstituted or substituted by aryl, cyanophenyl, heterocycle, C3-C10 cycloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, perfluoroalkyl, F, Cl, Br, RlOO-, Rl lS(0) m -, R l0c(
  • R9 is selected from: a) hydrogen, b) alkenyl, alkynyl, perfluoroalkyl, F, Cl, Br, R ' OO-, Rl lS(0) m -, Rl0C(O)NRl0-, (R10) 2 NC(0)-, Rl0 2 N- C(NRlO)-, CN, N02, RlOC(O)-, N3, -N(Rl ) 2 , or Rl lOC(O)NRl0-, and c) C1-C6 alkyl unsubstituted or substituted by perfluoroalkyl, F, Cl, Br, RlOO-, Rl lS(0) m -, R 10 C(O)NRl -, (RlO) 2 NC(0)-, Rl ⁇ 2N-C(NR O)-, CN, RlOC(O)-, N3, -N(RlO)2, or Rl l ⁇ C(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
  • 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 A2 is S(0)ml W is a heterocycle;
  • Z is selected from:
  • a unsubstituted or substituted group selected from aryl, heteroaryl, arylmethyl, heteroarylmethyl, arylsulfonyl, heteroaryl sulfonyl, wherein the substituted group is substituted with one or more of the following: a) Cl -4 alkyl, unsubstituted or substituted with: Cl-4 alkoxy, NR 6 R 7 , C3-6 cycloalkyl, aryl, heterocycle, HO, -S(0) m R 6a , or -C(0)NR6R7, b) aryl or heterocycle, c) halogen, d) OR6, e) NR6R7, f) CN, g) N02,
  • Rla is independently selected from: hydrogen or C1 -C6 alkyl
  • Rib is independently selected from: a) hydrogen, b) aryl, heterocycle, cycloalkyl, RlOO-, -N(RlO)2 or C2-C6 alkenyl, c) unsubstituted or substituted Cl-C6 alkyl wherein the substitutent on the substituted Cl-C6 alkyl is selected from unsubstituted or substituted aryl, heterocycle, cycloalkyl, alkenyl, RlOO- and -N(RlO)2; R3 and R ⁇ are independently selected from H and CH3;
  • R and R4 are independently selected from H; O or Ci -5 alkyl, unbranched or branched, unsubstituted or substituted with one or more of:
  • R6, R7 and R7a are independently selected from:
  • R6 is selected from: Cl-4 alkyl or C3-6 cycloalkyl, unsubstituted or substituted with: a) Cl -4 alkoxy, b) halogen, or c) aryl or heterocycle;
  • R8 is independently selected from: a) hydrogen, b) C1 -C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1 -C6 perfluoroalkyl, F, Cl, RK>0-, R 10C(O)NR 10-, CN, N02, (RlO)2N-C(NR lO)-, R lOC(O)-, -N(R lO)2, or
  • Rl l ⁇ C(O)NR l0- and c) C1-C6 alkyl substituted by C1-C6 perfluoroalkyl, Rl°0-, Rl C(0)NRlO-, (RlO) 2 N-C(NRlO)-, RlOC(O)-, -N(RlO)2, or Rl l ⁇ C(O)NRl0-;
  • R9 is selected from: a) hydrogen, b) C2-C6 alkenyl, C2-C6 alkynyl, C1 -C6 perfluoroalkyl, F, Cl, RlOO-, Rl lS(0) m -, R I 0 C(O)NRl0-, CN, N ⁇ 2, (RlO)2N-C(NRlO)-, RlOC(O)-, -N(RlO)2, or Rl l ⁇ C(O)NRl0-, and c) Cl -C6 alkyl unsubstituted or substituted by Cl -C6 perfluoroalkyl, F, Cl, RlOO-, Rl lS(0) ⁇ r, R 10 C(O)NRl0-, CN, (RlO)2N-C(NRlO)-, R10C(O)-, -N(RlO)2, or Rl l ⁇ C(O)NRl0-;
  • RlO is independently selected from hydrogen, C1-C6 alkyl, benzyl and aryl;
  • Rl 1 is independently selected from Cl -C 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 l 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 selected from:
  • a unsubstituted or substituted group selected from aryl, heteroaryl, arylmethyl, heteroarylmethyl, arylsulfonyl, heteroarylsulfonyl, wherein the substituted group is substituted with one or more of the following: a) Cl -4 alkyl, unsubstituted or substituted with:
  • C3-C6 cycloalkyl is substituted with one or two of the following: a) Cl-4 alkoxy, b) NR6R7,
  • Rla is selected from: hydrogen or C1 -C6 alkyl
  • Rib is independently selected from: a) hydrogen, b) aryl, heterocycle, cycloalkyl, RlOO-, -N(Rl O)2 or C2-C6 alkenyl, c) C1-C6 alkyl unsubstituted or substituted by aryl, heterocycle, cycloalkyl, alkenyl, Rl ⁇ O-, or -N(RlO)2;
  • R3 and R ⁇ are independently selected from H and CH3;
  • NR 6 R 7 R2 and R4 are independently selected from H; O or C 1-5 alkyl, unbranched or branched, unsubstituted or substituted with one or more of:
  • R6 and R7 are independently selected from: a) hydrogen, b) Cl -C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, Cl -C6 perfluoroalkyl, F, Cl, RlOO-, R!0C(O)NR10-, CN, N02, (RlO)2N-C(NRlO)-, RlOC(O)-, RlO ⁇ C(O)-, -N(RlO)2, or RllOC(O)NRl -,and c) C1-C6 alkyl substituted by C1-C6 perfluoroalkyl, RlOO-,
  • R8 is independently selected from: a) hydrogen, b) C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 perfluoroalkyl, F, Cl, R OO-, R10C(O)NR10-, CN, N02, (RlO)2N-C(NRlO)-, RlOc(O)-, -N(Rl°)2, or Rll ⁇ C(O)NRl0-, and c) C 1 -C6 alky 1 substituted by C l -C6 perfluoroalkyl , R 1 °0- , Rl0C(O)NR l -, (RlO ⁇ NRl )-, RlOc(O)-,
  • R is hydrogen or methyl
  • RIO is independently selected from hydrogen, Cl -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) C 1 -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 ;
  • X is -CH2-
  • Z is selected from:
  • a unsubstituted or substituted group selected from aryl, heteroaryl, arylmethyl, heteroarylmethyl, arylsulfonyl, heteroarylsulfonyl, wherein the substituted group is substituted with one or more of the following: a) Cl -4 alkyl, unsubstituted or substituted with: Cl-4 alkoxy, NR 6 R 7 , C3-6 cycloalkyl, aryl, heterocycle, HO, -S(0)mR6a, 0 r -C(0)NR6R7, b) aryl or heterocycle, c) halogen, d) OR 6 ' e) NR6R7,
  • 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
  • Rib is independently selected from: a) hydrogen, b) aryl, heterocycle, cycloalkyl, Rl°0-, -N(R J 0) 2 or C2-C6 alkenyl, c) Cl-C6 alkyl unsubstituted or substituted by aryl, heterocycle, cycloalkyl, alkenyl, RlOO-, or -N(R 10) 2;
  • R3 and R ⁇ are independently selected from H and CH3;
  • R2 and R4 are independently selected from H; O or C 1 -5 alkyl, unbranched or branched, unsubstituted or substituted with one or more of: 1 ) aryl, 2) heterocycle,
  • R 6 and R are independently selected from: a) hydrogen, b) C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 perfluoroalkyl, F, Cl, RlOO-, R!0C(O)NR1 -, CN, NO2, (Rl )2N-C(NRl )-, RlOc(O)-, RlO ⁇ C(O)-, -N(Rl )2, or RHOC(O)NR10-, and c) C l -C6 alkyl substituted by C 1 -C6 perfluoroalkyl , R °0-, R!0C(O)NR10-, (R10) 2 N-C(NR10)-, RlOc(O)-, Rl ⁇ C(O)-, -N(RlO) , or Rl l ⁇ C(O)NRl0- ;
  • 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, (R10)2N-C(NR10)-, RlOc(O)-, -N(RlO)2, or RHOC(O)NR10-, and c) C1-C6 alkyl substituted by C1-C6 perfluoroalkyl, Rl°0-, R!0C(O)NR10-, (RlO)2N-C(NRlO)-, RlOC(O)-, -N(RlO)2, or Rl l ⁇ C(O)NRl0- ;
  • RlO is independently selected from hydrogen, -C6 alkyl, benzyl and aryl;
  • Rl 1 is independently selected from C1-C6 alkyl and aryl
  • Z is selected from:
  • a unsubstituted or substituted group selected from aryl, heteroaryl, arylmethyl, heteroarylmethyl, arylsulfonyl, heteroarylsulfonyl, wherein the substituted group is substituted with one or more of the following: a) Cl-4 alkyl, unsubstituted or substituted with: Cl-4 alkoxy, NR 6 R 7 , C3-6 cycloalkyl, aryl, heterocycle, HO, -S(0) m R6a, or -C(0)NR6R7, b) aryl or heterocycle, c) halogen, d) OR6, e) NR6R , f) CN, g) N02, h) CF3; i) -S(0) m R6a, j) -C(0)NR6R7, or k) C3-C6 cycloalkyl; or
  • Rib is independently selected from: a) hydrogen, b) aryl, heterocycle, cycloalkyl, RlOO-, -N(R1°)2 or C2-C6 alkenyl, c) C1-C6 alkyl unsubstituted or substituted by aryl, heterocycle, cycloalkyl, alkenyl, Rl O-, or -N(RlO)2;
  • R2 and R4 are independently selected from: hydrogen or -C6 alkyl
  • R3 and R5 are hydrogen
  • R 6 and R are independently selected from: a) hydrogen, b) C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 perfluoroalkyl, F, Cl, Rl°0-, R!0C(O)NR10-, CN, N02, (Rl )2N-C(NRlO)-, RlOc(O)-, RlO ⁇ C(O)-, -N(RlO)2, or RH ⁇ C(O)NRl0-,and c) C1-C6 alkyl substituted by C1-C6 perfluoroalkyl, RlOO-, Rl C(0)NRlO-, (R10) 2 N-C(NR10)-, RlOc(O)-, RlO ⁇ C(O)-, -N(RlO)2, or Rl 1OC(O)NR10- ;
  • Rl is independently selected from hydrogen, C1-C6 alkyl, benzyl and aryl;
  • Rl 1 is independently selected from C1-C6 alkyl and aryl;
  • X is -CH2-;
  • the preferred compounds of this invention are as follows: 4-f 1 -(4-Cyanobenzyl)-5-imidazolylmethylJ- 1 -(3-chlorophenyl)- piperazine-2,5-dione
  • 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.
  • any variable e.g. aryl, heterocycle, Rl , R2 etc.
  • its definition on each occurence is independent at every other occurence.
  • combinations of substituents/or variables are permissible only if such combinations result in stable compounds.
  • alkyl is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms; “alkoxy” represents an alkyl group of indicated number of carbon atoms attached through an oxygen bridge.
  • Halogen or “halo” as used herein means fluoro, chloro, bromo and iodo.
  • aryl is intended to mean any stable monocyclic or bicyclic carbon ring of up to 7 members in each ring, wherein at least one ring is aromatic.
  • aryl elements include phenyl, naphthyl, tetrahydronaphthyl, indanyl, biphenyl, phenanthryl, anthryl or acenaphthyl.
  • heterocycle or heterocyclic represents a stable 5- to 7-membered monocyclic or stable 8- to 11 - membered bicyclic heterocyclic 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 stmcture.
  • heterocyclic elements include, but are not limited to, azepinyl, benzimidazolyl, benzisoxazolyl, benzofurazanyl, benzopyranyl, benzothiopyranyl, benzofuryl, benzothiazolyl, benzothienyl, benzoxazolyl, chromanyl, cinnolinyl, dihydrobenzofuryl, dihydrobenzothienyl, dihydrobenzothiopyranyl, dihydrobenzothiopyranyl sulfone, furyl, imidazolidinyl, imidazolinyl, imidazolyl, indolinyl, indolyl, isochromanyl, isoindolinyl, isoquinolinyl, isothiazolidinyl, isothiazolyl, isothiazolidinyl, morpholinyl, naphthyridinyl, oxadiazolyl,
  • heteroaryl is intended to mean any stable monocyclic or bicyclic carbon ring of up to 7 members in each ring, wherein at least one ring is aromatic and wherein from one to four carbon atoms are replaced by heteroatoms selected from the group consisting of N, O, and S.
  • heterocyclic elements include, but are not limited to, benzimidazolyl, benzisoxazolyl, benzofurazanyl, benzopyranyl, benzothiopyranyl, benzofuryl, benzothiazolyl, benzothienyl, benzoxazolyl, chromanyl, cinnolinyl, dihydrobenzofuryl, dihydrobenzothienyl, dihydrobenzothiopyranyl, dihydrobenzothiopyranyl sulfone, furyl, imidazolyl, indolinyl, indolyl, isochromanyl, isoindolinyl, isoquinolinyl, isothiazolyl, naphthyridinyl, oxadiazolyl, pyridyl, pyrazinyl, pyrazolyl, pyridazinyl, pyrimidinyl, pyrrolyl, quinazolin
  • the substituted group intended to mean a substituted Cl -8 alkyl, substituted C2-8 alkenyl, substituted C2-8 alkynyl, substituted aryl or substituted heterocycle from which the substitutent(s) R and R ⁇ are selected.
  • R 6 R a ?
  • R7 anc j R7a the substituted Cl -8 alkyl, substituted C3-6 cycloalkyl, substituted aroyl, substituted aryl, substituted heteroaroyl, substituted arylsulfonyl, substituted heteroarylsulfonyl and substituted heterocycle include moieties containing from 1 to 3 substitutents in addition to the point of attachment to the rest of the compound.
  • cyclic moieties When R and R ⁇ are combined to form - (CH2)u -, cyclic moieties are formed. Examples of such 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:
  • Rl and Rib are independently selected from: hydrogen, -N(R 10 )2, Rl0C(O)NRl0- or unsubstituted or substituted C1 -C6 alkyl wherein the substituent on the substituted Cl-C6 alkyl is selected from unsubstituted or substituted phenyl, -N(Rl ) 2 , R l O- and Rl0c(O)NRl0-.
  • R2 is selected from: H,
  • R3 is selected from: hydrogen and Cl -C6 alkyl.
  • R4 and R ⁇ are hydrogen.
  • R6, R7 and R a is selected from: hydrogen, unsubstituted or substituted -C6 alkyl, unsubstituted or substituted aryl and unsubstituted or substituted cycloalkyl.
  • R6a is unsubstituted or substituted Cl-C ⁇ alkyl, unsubstituted or substituted aryl and unsubstituted or substituted 10 cycloalkyl.
  • R9 is hydrogen or methyl. Most preferably, R is hydrogen.
  • R l° is selected from H, Cl -C6 alkyl and benzyl.
  • a 1 and A are independently selected from: a bond, -C(O)NR l0-, -NR IOC(O)-, O, -N(R 10)-, -S(0)2N(R 10)- a nd-
  • V is selected from hydrogen, heterocycle and aryl. More preferably, V is phenyl. 20
  • Z is selected from unsubstituted or substituted phenyl, unsubstituted or substituted naphthyl, unsubstituted or substituted pyridyl, unsubstituted or substituted furanyl, unsubstituted or substituted thienyl and unsubstituted or substituted alkyl. More preferably, Z is unsubstituted or substituted phenyl.
  • W is selected from imidazolinyl, imidazolyl, oxazolyl, pyrazolyl, pyyrohdinyl, thiazolyl and pyridyl. More preferably, W is selected from imidazolyl and pyridyl.
  • n and r are independently 0, 1 , or 2.
  • p is 1 , 2 or 3.
  • s is 0.
  • t is 1.
  • X is -CH2-.
  • any substituent or variable e.g., Rla, R , n, etc.
  • -N(R l )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, from readily available starting materials.
  • 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, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxy-benzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, trifluoroacetic and the like.
  • 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 either 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.
  • Reactions used to generate the compounds of this invention are prepared by employing reactions as shown in the Schemes 1-14, in addition to other standard manipulations such as ester hydrolysis, cleavage of protecting groups, etc., as may be known in the literature or exemplified in the experimental procedures.
  • the intermediate IV can be cyclized and reductively alkylated with a variety of aldehydes, such as IX.
  • 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 (Scheme 2).
  • 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 X can be deprotected to give the final compounds XI with trifluoroacetic acid in methylene chloride.
  • the final product XI is isolated in the salt form, for example, as a trifluoroacetate, hydrochloride or acetate salt, among others.
  • the product diamine XI can further be selectively protected to obtain XII, which can subsequently be reductively alkylated with a second aldehyde to obtain XIII. Removal of the protecting group, and conversion to cyclized products such as the dihydroimidazole XV can be accomplished by literature procedures.
  • the imidazole acetic acid XVI can be converted to the acetate XVII by standard procedures, and XVII can be first reacted with an alkyl halide, then treated with refluxing methanol to provide the regiospecifically alkylated imidazole acetic acid ester XIX.
  • the ester is hydrolyzed and the acid converted to the acid chloride.
  • Reaction with the suitably substituted lithium diketopiperazine XXI in the presence of condensing reagents such as 1 -(3- dimethylaminopropyl)-3-ethylcarbodiimide (EDC) leads to acylated products such as XXII.
  • Scheme 3a illustrates an alternative preparation of the instant compounds which incorporate the preferred benzylimidazolyl moiety.
  • cyclization affords the key intermediate l -(l-benzylimidazol-5-yl)piperazine-2,5-dione, which can then be reacted with a suitably substituted triaryl bismuth reagent or other suitable electrophiles (Scheme 3b).
  • the intermediate IV is cyclized/reductively alkylated with an aldehyde which also has a protected hydroxyl group, such as XXIII in Scheme 4, the protecting groups can be subsequently removed to unmask the hydroxyl group (Schemes 4, 5).
  • the alcohol can be oxidized under standard conditions to e.g.
  • the fully deprotected amino alcohol XXV can be reductively alkylated (under conditions described previously) with a variety of aldehydes to obtain secondary amines, such as XXIX (Scheme 5), or tertiary amines.
  • the Boc protected amino alcohol XXV can also be utilized to synthesize 4-(2-aziridinylmethyl)-2,5-diketopiperazines such as XXX (Scheme 6).
  • the intermediate IV can be reacted with aldehydes derived from amino acids such as O-alkylated tyrosines, according to standard procedures, to obtain compounds such as XXXV as shown in Scheme 7.
  • R' is an aryl group
  • XXXV can first be hydrogenated to unmask the phenol, and the amine group deprotected with acid to produce XXXVI.
  • the amine protecting group in XXXV can be removed, and O-alkylated phenolic amines such as XXXVII produced.
  • Reaction Scheme 8 provides an illustrative example the synthesis of compounds of the instant invention wherein the substituents R4 and R ⁇ are combined to form - (CH2)u -•
  • substituents R4 and R ⁇ are combined to form - (CH2)u -•
  • 1- aminocyclohexane-1 -carboxylic acid XXXVIII can be converted to the spirodiketopiperazine an intermediate IXL essentially according to the procedures outlined in Schemes 1 and 2.
  • the intermediate IXL can be deprotected as before, and carried on to final products as described in Schemes 3-8. It is understood that reagents utilized to provide the imidazolylalkyl substituent may be readily replaced by other reagents well known in the art and readily available to provide other N- substituents on the diketopiperazine.
  • Scheme 9 illustrates the use of an optionally substituted homoserine lactone XLII to prepare a Boc-protected intermediate XLIII.
  • Intermediate XLIII may be deprotected and cyclized/reductively alkylated or acylated as illustrated in the previous Schemes.
  • diketopiperazine XLIV may be mesylated and displaced by a suitable nucleophile, such as the sodium salt of ethane thiol, to provide an the sulfide XLV.
  • Diketopiperazine XLIV may also be oxidized to provide the carboxylic acid on diketopiperazine XLVI, which can be further utilized to form an ester or amide moiety.
  • Amino acids of the general formula XLVIII which have a sidechain not found in natural amino acids may be prepared by the reactions illustrated in Scheme 10 starting with the readily prepared imine XLIX.
  • 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 activity (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 al.FASEB Journal, 2:A3160 ( 1988)).
  • the instant compounds may also be useful for the treatment of fungal infections.
  • 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 i otonic.
  • the compounds of the instant invention may also be co- administered with other well known therapeutic agents that are selected for their particular usefulness against the condition that is being treated.
  • the instant compounds may be useful in combination with known anti -cancer and cy to toxic agents.
  • the instant compounds may be useful in combination with agents that are effective in the treatment and prevention of NF- 1 , restinosis, polycystic kidney disease, infections of hepatitis delta and related viruses and fungal infections.
  • compositions of this invention include aqueous solutions comprising compounds of this invention and pharmacolo- gically acceptable carriers, e.g., saline, at a pH level, e.g., 7.4. The solutions may be introduced into a patient's 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 60 mg/kg of body weight per day, preferably of between 0.5 mg kg of body weight to about 40 mg/kg of body weight per day.
  • the 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 sufficiently potent inhibitor i.e., one that has a Ki substantially smaller than the concentration of enzyme in the assay vessel
  • concentration of a sufficiently potent inhibitor i.e., one that has a Ki substantially smaller than the concentration of enzyme in the assay vessel
  • Chloride salts were obtained by passing an aqueous solution of the trifluoroacetic acid salt through a Biorad AG® 3X4 ion exchange resin column (100-200 mesh, Cl -form). Purification by HPLC was utilized for each of the Examples 1 , and 49 as set forth below.
  • Step A Preparation of l -triphenylmethyl-4-(hydroxymethyI)- imidazole
  • Step B Preparation of l -triphenylmethyl-4-(acetoxymethyl)- imidazole
  • Step C Preparation of l -(4-cyanobenzyl)-5-(acetoxymethyl)- imidazole hydrobromide
  • the filtrate was concentrated in vacuo to a volume 100 mL, reheated at 60 °C for another two hours, cooled to room temperature, and concentrated in vacuo to provide a pale yellow solid. All of the solid material was combined, dissolved in 500 mL of methanol, and warmed to 60 °C. After two hours, the solution was reconcentrated in vacuo to provide a white solid which was triturated with hexane to remove soluble materials. Removal of residual solvents in vacuo provided the titled product hydrobromide as a white solid (50.4 g, 67% yield, 89% purity by HPLC) which was used in the next step without further purification.
  • Step D Preparation of l -(4-cyanobenzyl)-5-(hydroxymethyl)- imidazole
  • Step E Preparation of l -(4-cyanobenzyl)-5-imidazole- carboxaldehyde
  • Step F Preparation of methyl 2-r(3-chlorophenyl)aminolacetate
  • Step G Preparation of 2-[(te/ -butoxycarbonyI)amino]-N-(3- chlorophen yl )-N- 1 (carbomethox v)meth yl 1 acetamide
  • Step H Preparation of 2-amino-N-(3-chlorophenyl)-/V- l(carbomethoxy)methyllacetamide
  • Step I Preparation of 4-[ 1 -(4-cyanobenzyl)-5-imidazolylmethyl ]-
  • the reaction is stirred for 12 hours, then concentrated in vacuo, and purified by silica gel chromatography.
  • the resulting product is taken up in CH2CI 2 and treated with excess 1 M HCl/ether solution, and concentrated in vacuo to provide the titled product hydrochloride.
  • the titled compound is prepared from the amine product from Step F of Example 1 using the reaction sequence described in Steps G, H, and I of Example 1 , except that in Step G the Boc-glycine is substituted with (S)-2-butoxycarbonylaminohexanoic acid.
  • the product is purified by silica gel chromatography, then taken up in CH 2 CI 2 and treated with excess 1 M HCl/ether solution, and concentrated in vacuo to provide the titled product hydrochloride.
  • Step B Preparation of 3-(4-cyanobenzyl)-4-(hydroxymethyl)- pyridine The title compound was obtained by sodium borohydride
  • Step C Preparation of 3-(4-cvanobenzyl)-4-pyridinal
  • the title compound was obtained by activated manganese dioxide (l .Og) oxidation of the alcohol from Step B (240 mg, 1.07 mmol) in dioxane (10 mL) at reflux for 30 min. Filtration and evaporation of the solvent provided title compound, mp 80-83°C.
  • Step D Preparation of l-(3-chlorophenyl)-4-[(3-(4- cyanobenzyl)pyridin-4-yl)methyl]-piperazine-2,5-dione hydrochloride
  • the titled compound is prepared from the pyridinal from Step C and the amine hydrochloride from Step H of Example 1 using the reductive alkylation procedured in Step I of Example 1.
  • the product is purified by silica gel chromatography, then taken up in CH 2 CI 2 and treated with excess 1 M HCl/ether solution, and concentrated in vacuo to provide the titled product hydrochloride.
  • Step A Preparation of l -(4-cyanobenzyl)-5-[N-
  • Step B Preparation of l-(4-cyanobenzyl)-5-[N-
  • Step C Preparation of l -[ l-(4-cyanobenzyl)-5- imidazolylmethyll-piperazine-2.5-dione Through a solution of Boc-protected amine from Step B
  • Step D Preparation of 4-[l -(4-cyanobenzyl)-5- imidazolylmethyl]- 1 -phenylpiperazine-2,5-dione hydrochloride
  • Bovine FPTase was assayed in a volume of 100 ⁇ l containing 100 mM N-(2- hydroxy ethyl) piperazine-N'-(2-ethane sulfonic acid) (HEPES), pH 7.4, 5 mM MgCl2, 5 mM dithiothreitol (DTT), 100 mM [ 3 H] -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 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 [ 3 H]-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 af, 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 nM 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
  • Examples 4 and 5 were tested for inhibitory activity against human FPTase by the assay described above and were found to have IC50 of ⁇ 50 ⁇ 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 51 :712-717, (1991). Cells in 10 cm dishes at 50-75% confluency are treated with the test compound (final concentration of solvent, methanol or dimethyl sulfoxide, is 0.1 %).
  • 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[ 5S]methionine (1000 Ci/mmol). After an additional 20 hours, the cells are lysed in 1 ml lysis buffer (1 % NP40/20 mM HEPES, pH 7.5/5 mM MgCl2/lmM DTT/10 mg/ml aprotinen/2 mg/ml leupeptin/2 mg/ml 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/lmM 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 nonfamesylated 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 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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Abstract

L'invention concerne des composés qui inhibent la farnésyl-protéine transférase (FTase) et la farnésylation de la protéine ras oncogène. L'invention a en outre pour objet des compositions chimiothérapeutiques contenant les composés de cette invention et des procédés pour inhiber la farnésyl-protéine transférase et la farnésylation de la protéine Ras oncogène.
EP97917599A 1996-04-03 1997-03-27 Inhibiteurs de farnesyl-proteine transferase Withdrawn EP0891349A4 (fr)

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WO1997036888A1 (fr) 1997-10-09
CA2249604A1 (fr) 1997-10-09
AU715667B2 (en) 2000-02-10
EP0891349A4 (fr) 2001-01-24
AU2587597A (en) 1997-10-22
JP2000507576A (ja) 2000-06-20

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