JP2001514191A - How to treat cancer - Google Patents

Abstract

  (57) [Summary] The present invention provides a method for inhibiting prenyl protein transferase and treating cancer, comprising administering to a mammal a prenyl protein transferase inhibitor that is an inhibitor of cellular processing of H-Ras and K4B-Ras proteins. provide. The invention particularly provides a method of inhibiting prenyl protein transferase and geranylgeranyl protein transferase type I by administering a compound that is a dual inhibitor of both of these prenyl protein transferases. The present invention provides for the convenient identification of compounds wherein the reading is a consequence of the biological activity of Ras-protein or of its activity, thereby inhibiting the cellular processing of H-Ras and K4B-Ras proteins. Methods for identifying such compounds are also provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

TECHNICAL FIELD The present invention relates to a method for inhibiting prenyl protein transferase utilizing a prenyl protein transferase that inhibits cell processing of both H-Ras protein and K4B-Ras protein, and a method for treating cancer. The invention also relates to a method for identifying such compounds.

BACKGROUND OF THE INVENTION Protein prenylation by intermediates in the isoprenoid biosynthetic pathway represents a group of post-translational modifications (Glomset, JA, Gelb, MH, and Farn).
swirth, C.I. C. (1990), Trends Biochem.
Sci. 15, Vol. 139-142; Maltese, W .; A. (1990
), FASEB J. 4, 3319-3328). This modification is typically required for membrane localization and function of these proteins. Prenylated proteins share a characteristic C-terminal sequence including CAAX (C, Cys; A, an aliphatic amino acid; X, another amino acid), XXCC or XCXC. C-terminal C
Three post-translational processing steps have been described for proteins with AAX sequences: the addition of 15 carbon (farnesyl) or 20 carbon (geranylgeranyl) isoprenoids to Cys residues, proteolytic resolution of the last three amino acids, and a new C -Methylation of terminal carboxylate (Cox, AD and Der
, C.I. J. (1992a), Critical Rev. Oncogenes
is Volume 3: 365-400; Newman, C.I. M. H. And Magee
, A. I. (1993), Biochim. Biophys. Acta 1
155: 79-96). Some proteins may have a fourth modification:
Or palmitoylation of two Cys residues N-terminal to a farnesylated Cys.
Although some mammalian cell proteins terminating in XCXC are carboxymethylated, it is not clear whether carboxymethylation occurs following prenylation of proteins terminating in the XXCC motif (Clarke, S. (1992) Year)
Authors, Annu. Rev .. Biochem. 61: 355-386). For all prenylated proteins, the addition of isoprenoids is the first step and is required for the next step (Cox, AD and Der, CJ (1992).
a), Critical Rev. Oncogenesis Volume 3: 36
5-400; Cox, A .; D. And Der, C.I. J. (1992b), C
current Opinion Cell Biol. Volume 4: 1008-10
16).

[0003] Three enzymes that catalyze the prenylation of proteins have been described: farnesyl protein transferase (FPTase), geranylgeranyl protein transferase type I (GGPTase-I) and geranylgeranyl protein transferase type II (GPTase-II, Rab GGPTase). Called). These enzymes are found in both yeast and mammalian cells (Clarke, 1992; Schafer, WR and Rine, J. Mol.
(1992), Annu. Rev .. Genet. Volume 30: 209-237
page). Each of these enzymes selectively uses farnesyl diphosphate or geranylgeranyl diphosphate as an isoprenoid donor to selectively identify protein substrates. FPTase farnesylates CAAX-containing proteins terminating in Ser, Met, Cys, Gln or Ala. For FPTase, C
AAX tetrapeptides contain the minimum region necessary for the interaction of protein substrates with enzymes. Enzymatic characterization of these three enzymes indicated that one could be selectively inhibited with little inhibitory effect on the other (M
oores, S.M. L. Schaber, M .; D. Mosser, S .; D. ,
Rands, E .; O'Hara, M .; B. Garsky, V .; M. , Mar
shall, M .; S. Pompliano, D .; L. And Gibbs, J. et al.
B. Author, J. et al. Biol. Chem. 266: 17438 (1991).
U.S. Pat. No. 5,470,832).

Prenylation reactions have been shown to be genetically essential for the function of various proteins (Clarke, 1992; Cox and Der, 1992a; Gibb).
s, J. et al. B. (1991), CEll; Vol. 65: pp. 1-4; Newman.
And Magee, 1993; Schafer and Rine, 1992)
. This need is such that CAAX Cy is such that the protein can no longer be prenylated.
It is often indicated by mutating the s receptor. The resulting protein lacks central biological activity. These studies show that genetically-defined "prenylation inhibitors can alter the physiological response regulated by prenylated proteins.
Provide proof of principle.

[0005] Ras proteins are part of a signaling pathway that couples cell surface growth factor receptors to nuclear signals that initiate cell proliferation. Biological and biochemical studies of Ras action indicate that Ras function is suitable for G-regulated proteins. In the inactive state, Ras is bound to GDP. Upon activation of the growth factor receptor,
Ras is triggered to replace GTP with GDP and change the conformation. GT
P-linked Ras proliferates the growth stimulating signal until the signal is stopped by Ras' intrinsic GTPase activity and the protein is returned to its inactive GDP-bound state (DR Lowy and DM Willumsen, Ann. .Re
v. Biochem. 62: 851-891 (1993)). Ras
Activates multiple intracellular signal transduction pathways, including the MAP Kinase and Rho / Rac pathways (Joneso)
n et al., Science 271: 810-812). One consequence of activation of the MAP kinase pathway is the activation of transcription factors such as Elk-1 and the transcription of specific proteins (R. Treisman, Current Opinio).
n in Genetics and Development (1994)
4: 96-101 and references therein).

[0006] Mutated ras genes are found in many human cancers, including colorectal cancer, exocrine pancreatic adenocarcinoma and myeloid leukemia. The protein products of these genes are GT
They lack Pase activity and essentially transmit growth stimulating signals.

[0007] Ras protein is one of several proteins known to carry out post-translational modifications. Farnesyl protein transferase covalently modifies the Cys thiol group of the RasCAAX box with a farnesyl group using farnesyl pyrophosphate (Reiss et al., Cell, 62: 81-88).
(1990); Schaber et al. Biol. Chem. 265th
Volume: 14701-14704 (1990); Schafer et al., Sci.
ence, 249: 1133-1139 (1990); Manne et al., Proc. Natl. Acad. Sci USA, Vol. 87: 7541-
7545 (1990)).

[0008] Ras must be localized to the plasma membrane for both normal and oncogene function. At least three post-translational modifications are associated with Ras membrane localization, and all three modifications occur at the C-terminus of Ras. RasC-terminal is “CAAX”
Or a sequence motif called the "Cys-Ass-Aaa-Xaa" box (Willumsen et al., Nature 310: 583-586 (1984)). This motif, depending on the particular sequence, serve as a signal sequence for the enzymes farnesyl protein transferase or geranylgeranyl protein transferase that catalyzes the alkylation of the cysteine residues of the CAAX motif with C ₁₅ or C ₂₀ isoprenoid, respectively (S .Cla
rke. Ann. Rev .. Biochem. Volume 61: Pages 355-386 (
1992)); R. Schaffer and J.M. Line, Ann. Rev
. Genetics 30: 209-237 (1992)).

Other farnesylated proteins include Ras-related GTP binding proteins such as RhoB, fungal mating factor, nuclear lamin, and the gamma subunit of transducin. James et al. Biol. Chem. Vol. 269: 14182 (
1994) identified a peroxisomal-associated protein, Pxf, which was also farnesylated. James et al. Also suggested that there are farnesylated proteins with unknown structure and function in addition to those listed above.

[0010] Inhibitors of farnesyl protein transferase (FPTase) have been described in two general classes. The first class includes analogs of farnesyl diphosphate (FPP), while the second class involves protein substrates (eg, Ras) for the enzyme. The peptide-induced inhibitors described are generally cysteine-containing molecules related to the CAAX motif, which is a signal for protein prenylation (Schaber et al., Ibid .; Reiss et al., Ibid .; Reiss.
Authors, PNAS, Vol. 88: 732-736 (1991). Such inhibitors may inhibit protein prenylation and serve as another substrate for the farnesyl protein transferase enzyme, or may be purely competitive inhibitors (US Pat. No. 5,141,851, University of Texas;
N. E. FIG. Kohl et al., Science, 260: 1934-1937 (1993); Graham et al. Med. Chem. , Vol. 37: 72
5 (1994)).

[0011] Mammalian cells express four types of Ras proteins (H-, N-, K4A- and K4B-Ras), of which K4B-Ras is Ra in human cancers.
It is the most frequently mutated form of s. The genes encoding these proteins are H-ras, N-ras, K4A-ras and K4B-Ras, respectively.
Is abbreviated. H-ras is an abbreviation for Harvey-ras. K4A-r
as and K4B-ras are abbreviations for Kirsten splice variants of ras containing the 4A and 4B exons, respectively. Inhibition of farnesyl protein transferase has been shown to inhibit the growth of H-ras-transformed cells in soft agar and modify other aspects of its transformed phenotype. Certain inhibitors of farnesyl protein transferase have also been shown to selectively inhibit the processing of H-ras oncoproteins intracellularly (NE Kohl et al.
Science, 260: 1934-1937 (1993) and G.
. L. James, et al., Science, 260: 1937-1942 (1993)). Recently, inhibitors of farnesyl protein transferase inhibit the growth of H-ras-dependent tumors in nude mice (NE Koh).
l Proc. Natl. Acad. Sci U. S. A. 91: 9
141-9145 (1994)), induces regression of mammary and salivary gland carcinomas in H-ras transgenic mice (NE Kohl et al., Nat.
ure Medicine, 1: 792-797 (1995)).

Indirect inhibition of farnesyl protein transferase in vivo has been demonstrated by lovastatin (Merck & Co., Lowway, NJ) and compactin (Hancock et al., Ibid .; Casey et al., Ibid .; Schafer
Et al., Science, Vol. 245: 379 (1989)). These agents inhibit HMG-CoA reductase and rate limiting enzymes for the production of polyisoprenoids include farnesyl pyrophosphate. HMG-Co
Inhibition of farnesyl pyrophosphate biosynthesis by inhibition of A reductase inhibits Ras membrane localization in cultured cells.

[0013] It has been disclosed that the lysine-rich region at the C-terminus of K4B-Ras and the terminal CVIM sequence confers resistance to inhibition of cellular processing of that protein by certain selective FPTase inhibitors (James et al.). , J. Biol.
. 270: 6221 (1995)). These FPTase inhibitors are:
It is effective in inhibiting the processing of H-Ras protein. James
Et al. Found that pre-nylation of K4B-Ras protein by GGTase resulted in selective FP
Suggested that it contributes to resistance to Tase inhibitors (Zhang et al., J. Bi.
ol. Chem. Volume 272: 10232 to 239 (1997); Row
ell et al. Biol. Chem. Volume 272: 14093-14097
Page (1997); Whyte et al. Biol. Chem. Vol. 272: 1
4459-14644 (1997)).

[0014] Several groups of scientists have recently disclosed compounds that are non-selective FPTase / GGTase inhibitors (Nagasu et al., Cancer Research).
55: 5310-5314 (1995); PCT application WO 9
5/25086).

Recently, assays useful for identifying inhibitors of FPTase that incorporate all or a portion of the K4B-Ras protein have been disclosed (PCT application WO 96/34113).
.

It is an object of the present invention to provide a prenyl protein transferase inhibitor comprising a HR
It is an object of the present invention to provide a method for inhibiting prenyl protein transferase, which utilizes a compound that inhibits cell processing of as and K4B-Ras proteins.

[0017] Compositions comprising such inhibitor compounds are also used in the present invention for treating cancer.

It is also an object of the present invention to provide a method for identifying a prenyl protein transferase inhibitor that is an inhibitor of the cellular processing of H-Ras and K4B-Ras proteins.

DISCLOSURE OF THE INVENTION The present invention inhibits prenyl protein transferase comprising administering to a mammal a prenyl protein transferase inhibitor that is an inhibitor of the cellular processing of H-Ras and K4B-Ras proteins. And a method of treating cancer. The invention particularly provides a method of inhibiting prenyl protein transferase and geranylgeranyl protein transferase type I by administering a compound that is a dual inhibitor of both of these prenyl protein transferases. The present invention provides that the reading is a result of the inhibition of the biological activity of Ras-protein or its activity, whereby H-Ras and K4B-R
Also provided are methods of identifying such compounds that provide for the convenient identification of compounds that inhibit cellular processing of as proteins.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for administering to a mammal in need thereof a pharmaceutically effective amount of a compound having certain characteristics that exhibits an in vivo effect as an inhibitor of cancer cell growth. And a method of inhibiting prenyl protein transferase. In particular the compounds are: a) 12 μl for K4B-Ras dependent activation of MAP kinase in cells
It is characterized by an inhibitory activity (IC ₅₀ ) lower than M.

Preferably, the compound utilized in the method comprises: a) 5 μM for K4B-Ras dependent activation of MAP kinase in cells
It is characterized by a lower inhibitory activity (IC ₅₀ ).

The compound is further characterized by one or more of the following features: b) Cells that are more than 5 times less inhibitory than Myr-Ras dependent activation of MAP kinase in cells (IC ₅₀ ). Kinase K4B-Ras in Tobacco
Inhibition of dependent activation activity _(IC 50); c) inhibitory activity against Myr-Ras dependent activation of MAP kinase in a cell (the MAP kinase at low cell 5 times more than the _{IC 50)} H- ras-C
Inhibitory activity (IC ₅₀ ) on VLL-dependent activation; d) More than 2-fold lower than the inhibitory activity (IC ₅₀ ) on H-ras-CVLL (SEQ ID NO: 1) -dependent activation of MPA kinase in cells Activity against H-Ras-dependent activation of MAP kinase in cells with a
C _50); d) constitutively in the pCMV-SEAP plasmid expressing SEAP protein in transfected cells in inhibitory activity on the expression of SEAP protein (the MAP kinase at low cell 5 times more than the _{IC 50)} H-ras-CVLL inhibitory activity _{(IC 50)} for dependent activation; and e) constitutively inhibit activity pCMV-SEAP plasmid expressing SEAP protein on the expression of SEAP protein in the transfected cells in _{(IC 50} ) Inhibitory activity (IC ₅₀ ) on K4B-Ras-dependent activation of MAP kinase in cells at least 5 times lower than that of ().

Preferably, the compound is further characterized by: c) an inhibitory activity (IC ₅₀ ) of less than 10 nM on H-Ras dependent activation of MAP kinase in cells.

Preferably, the prenyl protein transferase inhibited by the present method is both a farnesyl protein transferase and a geranylgeranyl protein transferase type I. Preferably, the compound administered is a dual inhibitor of farnesyl protein transferase and geranylgeranyl protein transferase type I.

[0025] Surprisingly, such effective dual inhibitors are useful for the in vivo development of cancer cells, particularly those characterized by mutated K4B-Ras proteins, at inhibitor concentrations that do not cause mechanistic toxicity. It has been found to be particularly useful as an inhibitor. The mechanistic toxicity of farnesyl protein transferase can be expected in rapidly growing tissues, such as bone marrow.

The present invention further relates to a method of identifying a prenyl protein transferase inhibitor that is effective in vivo as an inhibitor of cancer cell growth. The method provides a novel in vitro assay in which the readout is a result of the biological activity of Ras protein (or its inhibition) rather than a determination of the physical state of the Ras protein (whether or not the protein is processed) ( (Described in detail below). This assay differs from the previously disclosed assays that measure the degree of inhibition of Ras processing in cells. This is because the determination of the degree of processing can be performed using a high-throughput fluorometer and does not depend on the time-consuming use of polyacrylamide gel electrophoresis. Compounds that inhibit the processing of Ras protein, but not its biological activity, may be inappropriately identified by the Ras processing in cell assays disclosed above, which simply measure the extent to which the protein is processed. is there.

The assays useful in identifying prenyl protein transferase inhibitors of the invention include the following: a) Co-plasmid cells with an expression plasmid for the ras gene and an expression plasmid for the reporter construct encoding the product of the reporter gene. Transferring, b) incubating the cells in the presence of the test compound, and c) analyzing the lysate of the assay medium or cells for the presence of the product of the reporter gene.

Preferably, the product of the reporter gene is secreted alkaline phosphatase. When secreted alkaline phosphatase is used as a reporter,
The assay is called a SEAP assay. In the method described below, SEAP
The use of the assay is described. However, one of ordinary skill in the art will readily appreciate that other reporter systems including, but not limited to, luciferase, β-galactosidase, chloramphenicol acetyltransferase, and β-glucuronidase can be utilized.

[0029] Preferably, the expression of the reporter gene is controlled by a transcription factor activated by MAP kinase. The term MAP kinase includes, but is not limited to, ERK-1 (extracellularly regulated protein kinase), ERK-2, SAPK / JNK (stress activated protein kinase / C-jun N-terminal kinase) and p38.

[0030] Proteins incorporating the SEAP reporter structure are described in D.E. Defeo-Jone
S. et al., (Mol. Cell. Biol. 11: 2307-2310 (
1991)); E. FIG. Jones et al., (Oncogene. 6:74
5-5751 (1991)) and J. Org. Berger et al., (J. Biol.
. Chem. 263: 10016-10021 (1988)), but is not limited thereto. The SEAP reporter plasmids pDSE100 and pDSE101, described in Example 15 below, are also useful in the assays of the present invention. The SEAP reporter plasmid pCMV is also useful in the methods of the invention as a control plasmid to identify non-mechanistic toxicity of test compounds.

The term ras gene is used for H-ras, N-ras, K4B-ras, Myr
-Ras and H-ras-CVLL genes.

The expression plasmid for the ras gene of the present invention is pZIP-rasH, pZIP.
IP-rasN, pZIP-rasK4B, pSMS600, pSMS601,
pSMS620, pSMS621, pSMS622, pSMS630, pSMS
640, pSMS650, pBW1423 (BW Williamsem et al., Mol. Cell. Biol. 11: 6026-6033 (1991).
Year)), pRcCMV-H-ras-V12 and pRcCMV-H-ras-
v12, L189 (GL James, et al., J. Biol. Chem. 26th.
9: 27705-27714 (1994)).

Another expression vector that can be used to form an expression plasmid for the ras gene is pCl, pSI, pSport (Promega), p
BK-CMV, pBK-RSV (Stratagene), pEUK-C1 (
Clonetech), pCMV-L1C (Pharmingen) and p
cDNA 1.1 / Amp (manufactured by Invitrogen), but is not limited thereto.

[0034] The assay medium used in the present assay is selected from media useful for maintaining transiently transfected cells. Preferably, the medium lacks phenol red and is low in serum. Preferably, the assay medium is phenol red free DM
Contains EM, 2% mammalian serum, Pen / Strep, glutamine and non-essential amino acids (NEAA).

It is contemplated that virtually any commonly used transfectable host cell that is captured in a low serum growth medium can be used in connection with the present assay. Examples are typically used for eukaryotic expression such as C33a (ATCC: HTB-31), Rat1 and 3T3 cell lines. The preferred system for use in this assay turned out to be the human cell line C33a.

Preferably, the cancer cells are isolated after the expression plasmid has been co-transfected.

In a first embodiment of the invention, a method for identifying a prenyl protein transferase inhibitor comprises the steps of: a) evaluating a test compound in this assay wherein the ras gene is K-ras; b) determining whether the ras gene is Myr Assessing the test compound in the present assay which is -ras; and c) determining the activity of the test compound on Myr-Ras dependent activation of MAP kinase in the assay by K-Ras dependent activation of MAP kinase in the assay. Comparing the activity of the test compound to

Preferably, the K-ras gene used in the present method is a K4B-ras gene, but it is considered that the K4A-ras gene can also be used.

Preferably, the first embodiment of the method of the invention comprises one or both of the following additional steps: d) evaluating a test compound in the present assay wherein the ras gene is H-ras; and e) evaluating the test compound in the present assay wherein the ras gene is H-ras-CVLL.

In a second embodiment of the invention, a method for identifying a prenyl protein transferase inhibitor comprises the steps of: a) evaluating a test compound in this assay where the ras gene is H-Ras; -Evaluating the test compound in the present assay which is ras-CVLL; c) evaluating the test compound in the present assay wherein the ras gene is Myr-ras; and d) Myr-Ras dependency of MAP kinase in the present assay. Comparing the activity of the test compound to activation with the H-Ras-dependent activation of MAP kinase in the assay and the H-ras-CVLL-dependent activation of MAP kinase in the assay.

In a third embodiment of the invention, a method of identifying a prenyl protein transferase inhibitor comprises: a) evaluating a test compound in this assay where the Ras gene is N-Ras; b) determining whether the Ras gene is Myr Assessing the test compound in the assay which is -ras; and c) determining the activity of the test compound on Myr-Ras dependent activation of MAP kinase in the assay, N-Ras dependent activation of MAP kinase in the assay. Comparing the activity of the test compound to

In a fourth embodiment of the invention, a method of identifying a prenyl protein transferase inhibitor comprises the steps of: a) evaluating a test compound in this assay where the ras gene is H-Ras; Evaluating the test compound in the present assay which is -ras-CVLL; c) evaluating the test compound in the present assay wherein the cells have been transfected with the pCMV-SEAP plasmid in the absence of the ras gene transfection; and d) The activity of the test compound on H-Ras-CVLL-dependent activation of MAP kinase in this assay is determined by the MAP kinase H-Ras in this assay.
Comparing the activity of the test compound to dependent activation and the activity of the test compound to SEAP expression in step c) of the method.

Preferably, the assay utilized in the method for identifying an inhibitor is SEA
P assay.

Preferably, the pCMV-SEAP plasmid used in this assay is pCMV
V-SEAP-A plasmid.

The inhibitor compounds identified by the present methods are useful in inhibiting prenyl protein transferase and treating cancer and other proliferative diseases in mammals in need thereof. The above method of identifying a single compound that is a prenyl protein transferase inhibitor or a dual inhibitor of farnesyl protein transferase and geranylgeranyl protein transferase type I, comprises a selective farnesyl protein transferase inhibitor and a selective geranylgeranyl protein transferase type I inhibitor Can also be used to determine the optimal ratio of active ingredient in combination with

Preferably, the compounds of the present invention are MAP in cells in a SEAP assay.
Inhibitory concentrations (ICC) of less than 100 nM for H-Ras dependent activation of kinase ₅₀ ). More preferably, the compounds of the invention are used in SEAP assays.
Less than 10 nM for H-Ras dependent activation of MAP kinase in cells
Inhibitory concentration (IC₅₀). Preferably, for K-Ras4B dependent activation
Inhibitory activity (IC₅₀) And the inhibitory activity on H-Ras dependent activation
The ratio is lower than 2000.

Preferably, the compounds of the present invention are MAP in cells in a SEAP assay.
It has an inhibitory concentration (IC ₅₀ ) of less than 10 μM for H-Ras-CVLL-dependent activation of the kinase. More preferably, compounds of the invention have an inhibitory concentration (IC ₅₀ ) of less than 1 μM against H-Ras-CVLL-dependent activation of MAP kinase in cells in a SEAP assay. Preferably, H-Ras-
The ratio of the inhibitory activity (IC ₅₀ ) on CVLL-dependent activation to the inhibitory activity on H-Ras-dependent activation is about 2 to about 20,000. More preferably, H-
And Ras-CVLL dependent activation for inhibitory activity _(IC 50), the ratio of the inhibitory activity _{(IC 50)} with respect to H-Ras dependent activation is about 20 to about 2000.

Preferably, the compounds of the invention have an inhibitory concentration (IC ₅₀ ) of less than 5 μM on cellular N-Ras dependent activation of MAP kinase in a SEAP assay. More preferably, the compounds of the present invention have a low inhibitory concentration than 1μM _{(IC 5 0)} to the cell N-Ras dependent activation of MAP kinase in SEAP assay.

The compounds of the present invention selectively inhibit the processing of Ras protein and thus inhibit the growth of cells transformed by the ras oncogene. In the method of identifying a prenyl protein transferase inhibitor, the step of evaluating the activity of a test compound in this assay in which a SEAP plasmid is selected from a mutated ras gene designated as Myr-ras comprises: To inhibit the signal transduction independent of the inhibition of ATP. Because the mutated Myr-ras gene allows proteins to circumvent the requirement for prenylation for cellular activity (JE Buss et al., Science. 243:
1600 to 1603 (1989)). If close to the _{IC 50} of _{IC 50} of the test compound on cell Myr-Ras dependent activation of MAP kinase, the test compound on K4b-Ras dependent activation of MPA kinase in the same assay in the assay, inhibition of Ras prenylation It is difficult to determine the true effect of the test compound in Such inhibition is more non-specific cytotoxic than selective inhibition of Ras prenylation.

[0050] Non-specific cytotoxicity of a test compound can also be assessed by incubating cells transfected with the pCMV-SEAP plasmid and analyzing the assay medium for the presence of SEAP protein.

Thus, in one embodiment of the present invention, the MAP in a SEAP assay
And the activity of the test compound on cell Myr-Ras dependent activation of the kinase (as _{IC 5 0),} the ratio between the activity of the test compound on K4b-Ras dependent activation of MAP kinase in SEAP assay (as _{IC 50)} Preferably it is greater than 1. More preferably, MAP (measured in a SEAP assay)
Inhibitory activity (IC ₅₀ ) on Myr-Ras dependent activation of kinase and MA
The ratio of P kinase to inhibitory activity (IC ₅₀ ) on K4B-Ras-dependent activation is greater than 5.

“Inhibitory activity of MAP kinase on K4B-Ras-dependent activation of MAP kinase in cells (IC ₅₀ ) which is at least 5-fold lower than that on Myr-Ras-dependent activation of MAP kinase (IC ₅₀ ) ₅₀ ) "(and similar clauses for other comparators) is the" MAP kinase M (measured in the SEAP assay).
the ratio of the inhibitory activity (IC ₅₀ ) on yr-Ras dependent activation to the inhibitory activity (IC ₅₀ ) on K4B-Ras dependent activation of MAP kinase is greater than 5 ”(and other similar segments). ) Is understood to have the same meaning.

The activity of the test compound (as IC ₅₀ ) on cellular Myr-Ras dependent activation of MAP kinase in the SEAP assay and the M in the SEAP assay
Test compound activity on H-Ras-CVLL-dependent activation of AP kinase (
It is also preferred is greater than 1 the ratio between the IC _50). More preferably, (S
The ratio of the inhibitory activity of MAP kinase on Myr-Ras dependent activation of MAP kinase (measured by EAP assay) to the inhibitory activity of MAP kinase on H-Ras-CVLL dependent activation is greater than 5.

More preferably, the cell Myr- of MAP kinase in a SEAP assay.
And activity (as _{IC 50)} of the test compound on Ras dependent activation, SEAP
Ratio is greater than 5 and activity (as _{IC 50)} of the test compound on N-Ras dependent activation of MAP kinase in assays. Most preferably, (SEAP
The ratio of the inhibitory activity of MAP kinase on Myr-Ras dependent activation to the inhibitory activity of MAP kinase on N-Ras dependent activation (measured in the assay) is greater than 20.

In another embodiment of the present invention, the activity of the test compound on the expression of SEAP protein in cells transfected with the pCMV-SEAP plasmid (as an IC ₅₀ ) and the MAP kinase H-Ras- in SEAP assay. CVLL
Preferably, the ratio of the activity of the test compound to the dependent activation (as an IC ₅₀ ) is greater than 1. Most preferably, the inhibitory activity on the expression of SEAP protein pCMV-SEAP plasmid cells transfected with, and activity of the test compounds on H-Ras-CVLL dependent activation of MAP kinase in SEAP assay (as _{IC 50)} Is greater than 5.

When referring to a particular Ras protein here with terms such as “K4B-Ras”, “N-Ras”, “H-ras”, etc., such terms refer to the corresponding wild-type ras
Both proteins resulting from expression of the gene and various proteins resulting from expression of the ras gene, including various point mutations, are depicted. Here, "K4B-Ras", "NR"
When referring to a particular ras protein with terms such as "as", "H-ras" and the like, such terms refer to both the wild-type ras gene and the ras gene containing various point mutations.

The term prenyl protein transferase inhibiting compound means a gene encoding farnesyl protein transferase and geranylgeranyl protein transferase type I, or a compound that antagonizes, inhibits or counteracts the activity of the protein produced in response thereto.

The term selective as used herein refers to the inhibitory activity of a particular compound on one biological activity (such as inhibition of prenyl protein transferase) as the inhibitory activity of a compound on another biological activity. Show by comparison. It is understood that the greater the selectivity of a prenyl protein transferase inhibitor, the more such compounds will be favored by the treatment methods described.

For example, when describing a combination of a selective inhibitor of geranylgeranyl protein transferase type I and a farnesyl protein transferase, the in vitro activity assessed by the assay described in Example 11 A compound is considered a selective inhibitor of geranylgeranyl protein transferase type I if it is at least 10 times greater than the in vitro activity of the same compound on farnesyl protein transferase in the assay described in Example 10.
The compound has an in vitro farnesyl protein transferase inhibitory activity, as assessed, for example, by the assay described in Example 10 that is at least 10-fold greater than the in vitro activity of the same compound on geranylgeranyl protein transferase type I in the assay described in Example 11. If large, it is considered a selective inhibitor of farnesyl protein transferase. Preferably, the selective compound is at least 20% for one of the enzyme activities when comparing the geranylgeranyl protein transferase type I inhibitor to the farnesyl protein transferase inhibitor.
Shows twice the activity. More preferably, the selectivity is at least 100 times or more. It is understood that the greater the selectivity of a geranylgeranyl protein transferase type I inhibitor or farnesyl protein transferase inhibitor, the more such compounds will be preferred in such combinations.

A preferred therapeutic effect provided by the composition is the treatment of cancer, and in particular, the inhibition of the growth of cancerous tumors and / or the regression of cancerous tumors. Cancers that can be treated according to the invention described herein include brain, breast, colon, urogenital tract, prostate, skin, lymphatic system, pancreas, rectum, stomach, pharynx, liver and lung cancer. More specifically, such cancers include histiocytic lymphoma, lung adenocarcinoma, pancreatic cancer, colorectal cancer, small cell lung cancer, bladder cancer, head and neck cancer, acute and chronic leukemia, melanoma, and neuronal tumors. Including.

The compositions of the present invention are also useful for inhibiting other proliferative disorders, both benign and malignant, wherein Ras protein is abnormally activated as a result of cancerous mutations in other genes (ie, The ras gene itself is not activated by a sudden change to a cancerous state), and inhibition is achieved by administering an effective amount of the composition to a mammal in need of such treatment. For example, the compositions are useful in treating the benign proliferative disorder neurofibromatosis.

The compositions of the present invention are also useful for preventing restenosis after percutaneous transluminal coronary angioplasty by inhibiting intimal neoplasia (C. Indolfi et al., Na
cure Medicine, 1: 541-545 (1995).

The compositions may also be effective in treating and preventing polycystic kidney disease (D.
L. Schaffner et al., American Journal of Pa.
methodology, 142: 1051-1060 (1993) and B.
. Cowley, Jr et al., FASEB Journal, Volume 2: A3160
P. (1988)).

The compounds can also inhibit tumor angiogenesis and thereby affect tumor growth (J. Rak et al., Cancer Research, 55: 4).
575-4580 (1995)). Such anti-angiogenic properties of the compounds of the present invention may also be useful in the treatment of certain forms of visual impairment involving retinal neovascularization.

The compounds of the present invention are also useful in the treatment of certain viral infections, particularly in the treatment of hepatitis delta and related viruses (JS Glenn et al., Science).
ce, 256: 1331-1333 (1992)).

The compounds of the present invention are also useful in inhibiting the growth of vascular smooth muscle cells,
It may also be useful in the prevention and treatment of anterior sclerosis and diabetic vascular disorders.

The compounds of the present invention can be administered to a mammal, preferably a human, preferably alone or preferably in combination with a pharmaceutically acceptable carrier, excipient or diluent in a pharmaceutical composition, according to standard pharmaceutical practice. Can be administered. The compounds can be administered orally or parenterally, including intravenous, intramuscular, intraperitoneal, subcutaneous, rectal and topical routes of administration.

Pharmaceutical compositions containing the active ingredient may be in a form suitable for oral use, such as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or It may be syrup or elixir. Oral compositions can be prepared by any of the methods known in the art for the manufacture of pharmaceutical compositions, and such compositions provide pharmaceutically elegant and palatable formulations To this end, one or more reagents selected from the group consisting of sweeteners, flavors, colorants and preservatives can be included. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients are, for example, inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example microcrystalline cellulose, sodium croscarmellose.
binder, such as starch, gelatin, polyvinylpyrrolidone or gum arabic, and lubricants, such as magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to block the unpleasant taste of the drug or to delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained action over a longer period. For example, a water soluble taste masking material such as hydroxypropylmethyl cellulose or hydroxypropyl cellulose, or a time delay compound such as ethyl cellulose, cellulose acetate butyrate may be employed.

Formulations for oral use include hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin.
Alternatively, the active ingredient may be present as a soft gelatin capsule, mixed with a water-soluble carrier such as polyethylene glycol or an oil vehicle such as peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions.
Such excipients are suspending agents, for example, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, pyrivinylpyrrolidone, gum tragacanth and acacia; dispersing or wetting agents are naturally occurring phosphatides, such as lecithin, Or condensation products with alkylene oxide fatty acids, such as polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, such as heptadecaethylene-oxycetanol, or polyoxyethylene sorbitol monooleate. Condensation products of partial esters derived from fatty acids and hexitol with ethylene oxide, partial esters derived from fatty acids and hexitol anhydride and ethylene oxide Condensation products of Sid, for example, be a polyethylene sorbitan monooleate. The aqueous suspension can contain one or more preservatives, for example, ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and One or more sweeteners such as sucrose, saccharin or aspartame may be included.

Oily suspensions may be prepared by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as butylated hydroxyanisole or α-tocopherol.

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. provide. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, may also be present. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.

The pharmaceutical composition of the present invention may be in the form of an oil-in-water suspension. Oil phase is vegetable oil,
For example, it may be olive oil or peanut oil, or mineral oil such as liquid paraffin or mixtures thereof. Suitable emulsifiers are naturally occurring phosphatides, such as soy lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan monooleate, and condensation products of such partial esters with ethylene oxide, such as polyoxyethylene. It may be oxyethylene sorbitan monooleate. Emulsions may also contain sweetening, flavoring, preservative and antioxidant agents.

Syrups and elixirs can be prepared with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, flavoring and coloring agents and antioxidants.

The pharmaceutical compositions may be in the form of a sterile injectable aqueous solution. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.

The sterile injectable preparation may also be a sterile injectable oil-in-water microemulsion where the active ingredient is dissolved in the oily phase. For example, the active ingredient can first be dissolved in a mixture of soybean oil and lecithin. Next, the oil solution is introduced into a mixture of water and glycerol and processed to form a fine emulsion.

The injectable solutions or microemulsions can be introduced into a patient's blood stream by local bolus injection. It may also be advantageous to administer the solution or microemulsion in such a way as to maintain a constant circulating concentration of the compound. To maintain such a constant concentration, a continuous intravenous delivery device can be utilized. An example of such a device is the Deltec CADD-PLUS ^™ model 5400 intravenous pump.

The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension for intramuscular and subcutaneous administration. This suspension may be prepared according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation is a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example,
It may be a solution in 1,3-butanediol. In addition, sterile, fixed oils have conventionally been employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

The compounds of formula A may also be administered in the form of suppositories for rectal administration of the drug. These compositions are prepared by mixing the drug with a suitable nonirritating excipient which is solid at room temperature but liquid at rectal temperature, and thus melts in the rectum to release the drug. be able to. Such materials include cocoa butter, glycerinated gelatin, hydrogenated vegetable oils, mixtures of polyethylene glycols of various molecular weights and fatty acid esters of polyethylene glycol.

For topical use, creams, ointments, jellies, solutions or suspensions, etc., containing the compound of formula A are employed. (For the purpose of this application, topical application includes mouthwashes and gargles.) The compounds for the present invention may be administered intranasally by topical use of suitable intranasal vehicles and delivery devices, or by the transdermal route. It can be administered using a transdermal skin patch well known to those skilled in the art. For administration in a transdermal delivery system, the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.

As used herein, the term “composition” refers to a product comprising a specified amount of a specified component,
And, it is intended to include any product that results, directly or indirectly, from a specified amount of a specified combination of components.

The compounds identified by the present methods can also be administered with other well-known therapeutic agents, selected for their particular utility for the condition being treated.
For example, the compounds may be useful in combination with known anti-cancer and cytotoxic drugs. Similarly, the compounds may be useful in combination with agents that are effective in treating and preventing neurofibromatosis, restenosis, polycystic kidney disease, hepatitis delta infection, and related viral and fungal infections. The compounds may also be useful in combination with some other inhibitors of the signaling pathway that link the cell surface growth factor receptor to nuclear signal initiated cell proliferation.

The present compounds can be utilized in combination with a farnesyl pyrophosphate antagonistic inhibitor of the activity of farnesyl protein transferase or in combination with a compound having Raf antagonist activity. The compounds can also be administered together with compounds that are selective inhibitors of geranylgeranyl protein transferase or farnesyl protein transferase.

The compounds of the present invention may also be administered with other well-known cancer therapeutics, selected for their particular utility for the condition being treated. Such combinations of therapeutic agents include combinations of the present prenyl protein transferase inhibitors with antineoplastic agents. It is also understood that the present combinations of antineoplastic agents and inhibitors of prenyl protein transferase may be used in combination with other cancer and / or tumor treatment methods, including radiation therapy and surgery.

When prepared as a fixed dose, such combination products employ the combinations of the invention in the dosage ranges described below, and other pharmaceutically active agents within its approved dosage range. . The combinations of the present invention can also be combined sequentially with known pharmaceutically-acceptable agents if a combined combination formulation is inappropriate.

X delivered from an externally applied light beam or by implantation of a small radiation source
Radiation therapy, including radiation or gamma radiation, can also be used in combination with inhibitors of prenyl protein transferase alone for the treatment of cancer.

In addition, the compounds of the present invention may also be useful as radiosensitizers, as described in WO 97/38697, published October 23, 1997 and incorporated herein by reference.

The compounds may also be useful in combination with some other inhibitors of the signaling pathway that link the cell surface growth factor receptor to nuclear signal initiated cell proliferation. That is, the present compound can be used in combination with a farnesyl pyrophosphate antagonistic inhibitor of the activity of farnesyl protein transferase, or in combination with a compound having a Raf antagonistic activity.

This compound is disclosed in US Patent No. As described in 09 / 055,487, it may also be useful in combination with an integrin antagonist for the treatment of cancer.

As used herein, the term “integrin antagonist” refers to a compound that selectively antagonizes, inhibits or counteracts the binding of a physiological ligand to integrins involved in controlling angiogenesis or in tumor cell growth and invasion. Means In particular, the term refers to αvβ3 integrins and αvβ5 that selectively antagonize, inhibit or counteract the binding of a physiological ligand to αvβ3 integrin, or selectively antagonize, inhibit or counteract the binding of a physiological ligand to αvβ5 integrin. A compound that antagonizes, inhibits or counteracts the binding of a physiological ligand to both integrins, or antagonizes, inhibits or counteracts the activity of a particular integrin expressed on capillary endothelial cells. The terms include αvβ6, αvβ8, α1β1, α2β1, α
Antagonists of 5β1, α6β1 and α6β4 integrins are also meant. The terms are αvβ3, αvβ5, αvβ6, αvβ8, α1β1, α2β1, α5β1,
Antagonists of any combination of α6β1 and α6β4 integrins are also meant.
The compounds may also be useful in combination with other agents that inhibit angiogenesis, thereby inhibiting the growth and invasion of tumor cells, including but not limited to angiostatin and endostatin.

When the compositions of the present invention are administered to a human subject, the daily dosage will usually be determined by the prescribing physician, but dosages will generally be age, weight, and It depends on the response and the severity of the patient's symptoms.

In one application, a suitable amount of a prenyl protein transferase inhibitor is administered to a mammal undergoing treatment for cancer. Administration is 1k body weight per day
about 0.1 mg / g to about 60 mg / g, preferably about 0 / kg / day of body weight.
. It is performed in an amount of from 5 mg to about 40 mg of each inhibitor. Particular therapeutic dosages containing the present compositions comprise from about 0.01 mg to about 1000 mg of a prenyl protein transferase inhibitor. Preferably, the dosage comprises about 1 mg to about 1000 mg of prenyl protein transferase.

Examples of anti-tumor agents are typically microtubule stabilizers (paclitaxel (also known as Taxol®), docetaxel (also known as Taxotere®), or derivatives thereof; Alkylating agents, antimetabolites; epidophyllotoxins; antitumor enzymes; topoisomerase inhibitors; procarbazine; mitoxantrone; platinum coordination complexes; biological response modifiers and growth inhibitors; Includes therapeutics and hematopoietic growth factors.

Examples of anti-neoplastic agents include, for example, anthracyclines, vinca, mitomycin, bleomycin, cytotoxic nucleosides, taxanes, epothilones, discodermolide, pteridines, dainen, and podophyllotoxins. Among these particularly useful are, for example, doxorubicin, carminomycin, daunorubicin, aminopterin, methotrexate, metopterin, dichloromethotrexate, mitomycin C, porphyromycin, 5-fluorouracin, 6-mercaptopurine, gemcitabine, cytosine arabino Sido, podophyllotoxin or a podophyllotoxin derivative such as etoposide, etoposide phosphate or teniposide, melphalan, vinblastine, vincristine, leuclosidine,
Including vindesine, leurosin and paclitaxel. Other useful anti-neoplastic agents include estramustine, cisplatin, carboplatin, cyclophosphamide, bleomycin, gemcitibine, ifosamide, melphalan, hexamethylmelamine, thiotepa, cytarabine, idatrexate, trimetrexate, dacarbazine, L-asparaginase, Including camptothecin, CPT-11, topotecan, cytosine arabinoside, bicalutamide, flutamide, leuprolide, pyridobenzoindole derivatives, interferons and interleukins.

Compounds of the present invention identified by the properties set forth above include: (a) Compounds of Formula I:

[0096]

Embedded image Wherein R ^1a is selected from hydrogen or C ₁ -C ₆ alkyl; R ^1b is independently selected from a) to c) below: a) hydrogen b) aryl, heterocycle, cycloalkyl, R ¹⁰ O— , ^{-N (R} _{10) 2} or _C 2 -C ₆ alkenyl, c) aryl, heterocycle, cycloalkyl, ^{alkenyl, R} 10 O-or ^{-N (R} ₁₀₎ not been or substituted substituted by ₂ C ₁ -C ₆ alkyl; ^{R 3} and ^{R 4} are selected from H and CH _3; ^{R 2} is, H; unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl,

[0097]

Embedded imageOr one or more of unbranched or branched and unsubstituted or the following 1) to 5)
C substituted with_1-5Selected from alkyl: 1) aryl 2) heterocycle 3) OR⁶  4) SR^6a, SO₂R^6aOr 5)

[0098]

Embedded image And R²And R³Is optionally attached to the same carbon atom; R⁶And R⁷Is independently: H; substituted or substituted with the following a) to d)
Not C_1-4Alkyl, C_3-6From cycloalkyl, aryl, heterocycle
Selected: a) C_1-4Alkoxy, b) halogen, c) perfluoro-C_1-4Alkyl, or d) aryl or heterocycle; R^6aIs a C substituted or unsubstituted in a) to c) below._1-4Al
Kill or C_3-6Selected from cycloalkyl: a) C_1-4Alkoxy, b) halogen, or c) aryl or heterocycle; R⁸Is independently selected from a) to c) below: a) hydrogen, b) C_1-6Alkyl, C_2-6Alkenyl, C_2-6Alkynyl, C_{1 to 6} Perfluoroalkyl, F, Cl, R¹⁰O-, R¹⁰C (O) NR¹⁰−,
CN, NO₂, (R¹⁰)₂NC (NR¹⁰)-, R¹⁰C (O)-, -N (
R¹⁰)₂Or R¹¹OC (O) NR¹⁰-And c) C_1-6Perfluoroalkyl, R¹⁰O-, R¹⁰C (O) NR¹⁰ −, (R¹⁰)₂NC (NR¹⁰)-, R¹⁰C (O)-, -N (R¹⁰)₂ Or R¹¹OC (O) NR¹⁰C substituted by-_1-6Alkyl; R^9aRepresents hydrogen or methyl; R¹⁰Is independently hydrogen, C_1-6Alkyl, C_1-6Perfluoroalkyl
, 2,2,2-trifluoroethyl, benzyl and aryl; R¹¹Is independently C_1-6Selected from alkyl and aryl; A¹And A²Are independently: a bond, -CH = CH-, -C≡C-, -C (O
)-, -C (O) NR¹⁰-, O, -N (R¹⁰)-Or S (O)_mChoose from
V is selected from a) to e) below: a) hydrogen, b) pyrrolidinyl, imidazolyl, pyridinyl, thiazolyl, pyridonyl,
2-oxopiperidinyl, indolyl, quinolinyl, isoquinolinyl and thie
C) aryl, d) 0-4 carbon atoms are replaced by heteroatoms selected from O, S and N.
C₁~ C₂₀Alkyl, and e) C₂~ C₂₀Alkenyl, where A¹Is S (O)_mV is not hydrogen, but A¹V is hydrogen when is a bond
But n is 0, and A²Is S (O)_mX is -CH₂-Or -C (= O)-; Z is selected from the following 1) and 2): 1) aryl, heteroaryl, arylmethyl, heteroarylmethyl, a
Unsubstituted or substituted selected from reelsulfonyl and heteroarylsulfonyl
Wherein the substituted group is one or more of the following a) to k)
Substituted for: a) C_1-4Alkoxy, NR⁶R⁷, C_3-6Cycloalkyl, unsubstituted
Or substituted aryl, heterocycle, HO, -S (O)_mR^6aOr -C (O) NR ⁶ R⁷Substituted or unsubstituted C_1-4Alkyl, b) aryl or heterocycle, c) halogen, d) OR⁶, E) NR⁶R⁷, F) CN, g) NO₂H) CF₃, I) -S (O)_mR^6a, J) -C (O) NR⁶R⁷Or k) C₃~ C₆Cycloalkyl; or 2) unsubstituted C₁~ C₆Alkyl, substituted C₁~ C₆Alkyl, unsubstituted C₃~ C₆ Cycloalkyl or substituted C₃~ C₆Cycloalkyl, where substituted C₁~ C₆A
Alkyl and substitution C₃~ C₆Cycloalkyl is one or more of the following a) to h)
Substituted with at least one species: a) C_1-4Alkoxy, b) NR⁶R⁷C) C_3-6Cycloalkyl, d) -NR⁶C (O) R⁷, E) HO, f) -S (O)_mR^6aG) halogen; or h) perfluoroalkyl; m represents 0, 1 or 2; n represents 0, 1, 2, 3 or 4; And r represents 0 to 5, provided that when V is hydrogen, r represents 0; provided that the substituent (R⁸)_r-VA¹(CR^1a ₂)_nA²(CR^1a ₂)_n− Is
Not H; (b) inhibitors of farnesyl protein transferase represented by formula II:
:

[0099]

Embedded imageWhere R^1aIs hydrogen or C₁~ C₆R selected from alkyl;^1bIs independently selected from a) to c) below: a) hydrogen b) aryl, heterocycle, cycloalkyl, R¹⁰O-, -N (R¹⁰)₂ Or C₂~ C₆Alkenyl, c) unsubstituted or substituted aryl, heterocycle, cycloalkyl, alkenyl,
R¹⁰O- or -N (R¹⁰)₂C substituted or unsubstituted by ₁ ~ C₆Alkyl; R^1cIs selected from a) to c) below: a) hydrogen, b) unsubstituted or substituted C₁~ C₆Alkyl, where substituted C₁~ C₆Alkyl
The above substituents are unsubstituted or substituted aryl, heterocycle, C₃~ C₁₀Cycloal
Kill, C₂~ C₆Alkenyl, C₂~ C₆Alkynyl, R¹⁰O-, R¹¹S (
O)_m-, R¹⁰C (O) NR¹⁰−, (R¹⁰)₂NC (O)-, CN, (
R¹⁰)₂NC (NR¹⁰)-, R¹⁰C (O)-, R¹⁰OC (O)-, N ₃ , -N (R¹⁰)₂, And R¹¹OC (O) NR¹⁰-And
C) unsubstituted or substituted aryl;³And R⁴Is independently H and CH₃Selected from; R²Is H; OR¹⁰;

[0100]

Embedded image Or selected from unbranched or branched C _1-5 alkyl substituted or unsubstituted with one or more of the following 1) to 5): 1) aryl, 2) heterocycle, 3) OR ⁶ , 4) SR ^6a , SO ₂ R ^6a , or 5)

[0101]

Embedded image And R ² , R ³ and R ⁴ are optionally attached to the same carbon atom; R ⁶ and R ⁷ are independently H; C ₁ substituted or unsubstituted with a) to c) below. _{to 4 alkyl, C 3 to 6} cycloalkyl, aryl, heteroaryl ring: _{a) C 1 to 4} alkoxy, b) halogen, or c) aryl or heterocycle,; ^{R 6a} is below a) to c) R ⁸ is independently selected from substituted or unsubstituted C _1-4 alkyl or C _3-6 cycloalkyl: a) C _1-4 alkoxy, b) halogen, or c) aryl or heterocycle; Te, is selected from the following a) ~c): a) hydrogen, _{b) C 1 to 6 alkyl, C 2 to 6 alkenyl, C 2 to 6 alkynyl, C. 1 to 6} perfluoroalkyl, F, ^{Cl, R 10} O-, R ^{0 C (O) NR 10 -} ,
_{^{CN, NO 2, (R 10}} ) 2 N-C (NR 10) -, R 10 C (O) -, - N (
R ¹⁰⁾ ₂ or ^{R 11 OC (O) NR 10} -, and _{c) C 1 to 6} ^{perfluoroalkyl, R 10 O-, R 10 C} (O) NR 10 -, (R 10) 2 N-C ( ^{NR 10) -, R 10 C} (O) -, - N (R 10) 2 or ^{R 11 OC (O) NR 10} - which is substituted by _{C 1 to 6} alkyl; ^{R 9a} represents hydrogen or methyl; R ¹⁰ is independently selected from hydrogen, C _1-6 alkyl, C _1-6 perfluoroalkyl, 2,2,2-trifluoroethyl, benzyl and aryl; R ¹¹ is independently C ₁ It is selected from ₆ alkyl and aryl; R ¹² is, H; unsubstituted or substituted C _{1 to 8} alkyl is selected from unsubstituted or substituted aryl or unsubstituted or substituted heterocycle, wherein the substituted alkyl, substituted a The reel or substituted hetero ring is substituted with one or more of the following 1) to 18): 1) an aryl or hetero ring substituted or unsubstituted with the following a) to h), _{a) C 1 to 4 ^{alkyl, b) (CH 2) p}} oR 6, c) (CH 2) p NR 6 R 7, d) halogen, e) CN, f) aryl or heteroaryl, g) perfluoro - C _1-4 alkyl, h) SR ^6a , S (O) R ^6a , SO ₂ R ^a , 2) C _3-6 cycloalkyl, 3) OR ⁶ , 4) SR ^6a , S (O) R ^6a or SO _{^{2 a, 5) -NR 6 R}} 7,

[0102]

Embedded image 15) N₃, 16) F, 17) Perfluoro-C_1-4-Alkyl, or 18) C_1-6-Alkyl; A¹And A²Are independently a bond, -CH = CH-, -C≡C-, -C (O
)-, -C (O) NR¹⁰-, -NR¹⁰C (O)-, O, -N (R¹⁰)-,
Or S (O)_mV is selected from a) to e) below: a) hydrogen, b) pyrrolidinyl, imidazolyl, pyridinyl, thiazolyl, pyridonyl,
2-oxopiperidinyl, indolyl, quinolinyl, isoquinolinyl and thie
C) aryl, d) 0-4 carbon atoms are replaced by heteroatoms selected from O, S and N.
C₁~ C₂₀Alkyl, and e) C₂~ C₂₀Alkenyl, where A¹Is S (O)_mV is not hydrogen, but A¹V is hydrogen when is a bond
But n is 0, and A²Is S (O)_mX is -CH₂— Or —C (＝ O) —; X¹Is a bond, -C (= O)-, -NR⁶C (= O)-, -NR⁶-, -O-
Or -S (= O)_mY is selected from the following a) to c): a) hydrogen b) R¹⁰O-, R¹¹S (O)_m-, R¹⁰C (O) NR¹⁰−, (R^{1 0} )₂NC (O)-, CN, NO₂, (R¹⁰)₂NC (NR¹⁰)-, R ¹² C (O)-, R¹⁰OC (O)-, N₃, F, -N (R¹⁰)₂Or R ¹¹ OC (O) NR¹⁰-, C) unsubstituted or substituted C_1-6Alkyl, where substituted C_1-6On alkyl
The substituent is unsubstituted or substituted aryl, R¹⁰O-, R¹⁰C (O) NR¹⁰−,
(R¹⁰)₂NC (O)-, R¹⁰C (O)-and R¹⁰From OC (O)-
Z is an unsubstituted or substituted aryl, wherein the substituted aryl is any of the following 1) to 11)
Substituted with one or more kinds: 1) C substituted or unsubstituted with the following a) to g)_1-4Al
Kill, a) C_1-4Alkoxy, b) NR⁶R⁷C) C_3-6Cycloalkyl, d) aryl, substituted aryl or heterocycle, e) HO, f) -S (O)_mR^6aOr g) -C (O) NR⁶R⁷, 2) aryl or heterocycle, 3) halogen, 4) OR⁶, 5) NR⁶R⁷, 6) CN, 7) NO₂8) CF₃, 9) -S (O)_mR^6a, 10) -C (O) NR⁶R⁷Or 11) C₃~ C₆M represents 0, 1 or 2; n represents 0, 1, 2, 3 or 4; p represents 0, 1, 2, 3 or 4; 5 with the proviso that when V is hydrogen, r represents 0; and v represents 0, 1 or 2; (c) a compound of formula III

[0103]

Embedded image R¹Is independently hydrogen or C₁~ C₆R selected from alkyl;²Is independently selected from a) to c) below: a) hydrogen b) substituted or unsubstituted aryl, substituted or unsubstituted heterocycle, C₃~ C₁₀ Cycloalkyl, R¹⁰O- or C₂~ C₆Alkenyl, c) aryl, heterocycle, C₃~ C₁₀Cycloalkyl, C₂~ C₆Arche
Nil, R¹⁰O- or -N (R¹⁰)₂Substituted or unsubstituted
C₁~ C₆Alkyl; R³Is selected from the following a) to d): a) hydrogen, b) C₂~ C₆Alkenyl, R¹⁰O-, R¹¹S (O)_m-, R¹⁰C (
O) NR¹⁰-, CN, N₃, (R¹⁰)₂NC (NR¹⁰)-, R¹⁰C (
O)-, -N (R¹⁰)₂Or R¹¹OC (O) NR¹⁰Replaced by
Or unsubstituted C₁~ C₆Alkyl, c) substituted or unsubstituted aryl, substituted or unsubstituted heterocycle, C₃~ C₁₀ Cycloalkyl, C₂~ C₆Alkenyl, fluoro, chloro, R¹²O-, R^{1 1} S (O)_m-, R¹⁰C (O) NR¹⁰-, CN, NO₂, (R¹⁰)₂N-
C (NR¹⁰)-, R¹⁰C (O)-, N₃, -N (R¹⁰)₂Or R¹¹ OC (O) NR¹⁰-And d) substituted or unsubstituted aryl, substituted or unsubstituted heterocycle and C₃~ C ₁₀ C substituted with an unsubstituted or substituted group selected from cycloalkyl₁ ~ C₆Alkyl; R⁴And R⁵Is independently selected from a) to d) below: a) hydrogen, b) C₂~ C₆Alkenyl, R¹⁰O-, R¹¹S (O)_m-, R¹⁰C (
O) NR¹⁰-, CN, N₃, (R¹⁰)₂NC (NR¹⁰)-, R¹⁰C (
O)-, -N (R¹⁰)₂Or R¹¹OC (O) NR¹⁰Replaced by
Or unsubstituted C₁~ C₆Alkyl, c) substituted or unsubstituted aryl, substituted or unsubstituted heterocycle, C₃~ C₁₀ Cycloalkyl, C₂~ C₆Alkenyl, fluoro, chloro, R¹⁰O-, R^{1 1} S (O)_m-, R¹⁰C (O) NR¹⁰-, CN, NO₂, (R¹⁰)₂N-
C (NR¹⁰)-, R¹⁰C (O)-, N₃, -N (R¹⁰)₂Or R¹¹ OC (O) NR¹⁰-And d) substituted or unsubstituted aryl, substituted or unsubstituted heterocycle and C₃~ C ₁₀ C substituted with an unsubstituted or substituted group selected from cycloalkyl₁ ~ C₆Alkyl; R⁶Is independently selected from a) to c) below: a) hydrogen b) substituted or unsubstituted aryl, substituted or unsubstituted heterocycle, C_1-6Al
Kill, C_2-6Alkenyl, C_2-6Alkynyl, C_1-6Perfluoroalkyl
Le, F, Cl, R¹⁰O-, allyloxy, R¹⁰C (O) NR¹⁰-, CN, N
O₂, (R¹⁰)₂NC (NR¹⁰)-, R¹⁰C (O)-, -N (R¹⁰) ₂ , (R¹²)₂NC (O)-or R¹¹OC (O) NR¹⁰-And c) C_1-6Perfluoroalkyl, R¹⁰O-, R¹⁰C (O) NR¹⁰ −, (R¹⁰)₂NC (NR¹⁰)-, R¹⁰C (O), -N (R¹⁰)₂,
Or R¹¹OC (O) NR¹⁰C substituted with-₁~ C₆Alkyl; R⁷Is independently selected from a) to e) below: a) hydrogen, b) unsubstituted or substituted aryl, c) unsubstituted or substituted heterocycle, d) unsubstituted or substituted cycloalkyl, and e) hydrogen Or selected from aryl, heterocycle and cycloalkyl
C substituted with unsubstituted or substituted groups_1-6Alkyl; wherein the heterocycle is
Pyrrolidinyl, imidazolyl, pyridinyl, thiazolyl, pyridonyl, indori
R, quinolinyl, isoquinolinyl, and thienyl; R⁸Is selected from a) to c) below: a) hydrogen, b) C_1-6Alkyl, C_2-6Alkenyl, C_2-6Alkynyl, C_{1 to 6} Perfluoroalkyl, F, Cl, R¹⁰O-, R¹⁰C (O) NR¹⁰−,
CN, NO₂, (R¹⁰)₂NC (NR¹⁰)-, R¹⁰C (O)-, R¹⁰ OC (O)-, -N (R¹⁰)₂Or R¹¹OC (O) NR¹⁰-And c) C_1-6Perfluoroalkyl, R¹⁰O-, R¹⁰C (O) NR¹⁰ −, (R¹⁰)₂NC (NR¹⁰)-, R¹⁰C (O)-, R¹⁰OC (O)
-, -N (R¹⁰)₂Or R¹¹OC (O) NR¹⁰Replaced by-
C_1-6Alkyl; R⁹Is selected from a) to c) below: a) hydrogen, b) C_2-6Alkenyl, C_2-6Alkynyl, C_1-6Perfluoroal
Kill, F, Cl, R¹⁰O-, R¹¹S (O)_m-, R¹⁰C (O) NR¹⁰−
, CN, NO₂, (R¹⁰)₂NC (NR¹⁰)-, R¹⁰C (O)-, R^{1 0} OC (O)-, -N (R¹⁰)₂Or R¹¹OC (O) NR¹⁰−, And
C) C₁~ C₆Perfluoroalkyl, F, Cl, R¹⁰O-, R¹¹S (O) _m -, R¹⁰C (O) NR¹⁰−, CN, (R¹⁰)₂NC (NR¹⁰)-,
R¹⁰C (O)-, R¹⁰OC (O)-, -N (R¹⁰)₂Or R¹¹OC
(O) NR¹⁰C substituted or unsubstituted by-₁~ C₆Alkyl
R¹⁰Is independently hydrogen, C_1-6Alkyl, C_1-6Perfluoroalkyl
R, 2,2,2-trifluoroethyl, benzyl and aryl; R¹¹Is independently C_1-6Selected from alkyl and aryl; R¹²Is independently hydrogen, C₁~ C₆Alkyl, CO₂R¹⁰Replaced by
C₁~ C₆C substituted with alkyl or aryl₁~ C₆Alkyl, substituted aryl
C substituted with₁~ C₆Alkyl, heterocyclic substituted C₁~ C₆Alkyl,
C substituted with a substituted heterocycle₁~ C₆Alkyl, aryl and substituted aryl
Selected from: A¹And A²Are independently: a bond, -CH = CH-, -C≡C-, -C (O
)-, -C (O) NR⁷-, -NR⁷C (O)-, -S (O)₂NR⁷-, -N
R⁷S (O)₂-, O, -N (R⁷)-Or S (O)_mSelected from; A³Is a bond, -C (O) NR⁷-, -NR⁷C (O)-, -S (O)₂NR ⁷ -, -NR⁷S (O)₂-Or -N (R⁷)-; A⁴Is a bond, O, -N (R⁷V is selected from a) to e) below: a) hydrogen, b) pyrrolidinyl, imidazolyl, pyridinyl, thiazolyl, pyridonyl,
2-oxopiperidinyl, indolyl, quinolinyl, isoquinolinyl and thie
C) aryl, d) 0-4 carbon atoms are replaced by heteroatoms selected from O, S and N.
C₁~ C₂₀Alkyl, and e) C₂~ C₂₀Alkenyl, where A¹Is S (O)_mV is not hydrogen, but A¹V is hydrogen when is a bond
But n is 0, and A²Is S (O)_mZ is independently (R¹)₂Or represents O; m represents 0, 1 or 2; n represents 0, 1, 2, 3 or 4; p represents 0, 1, 2, 3 or 4; And r represents 0 to 5, provided that when V is hydrogen, r represents 0; (d) a compound represented by the following formula A:

[0104]

Embedded imageWhere R^1aIs hydrogen or C₁~ C₆R selected from alkyl;^1bIs independently selected from a) to c) below: a) hydrogen b) aryl, heterocycle, cycloalkyl, R¹⁰O-, -N (R¹⁰)₂ Or C₂~ C₆Alkenyl, c) aryl, heterocycle, cycloalkyl, alkenyl, R¹⁰O- or
−N (R¹⁰)₂C substituted or unsubstituted by₁~ C₆Alkyl
R^2a, R^2bAnd R³Is independently selected from a) to d) below: a) hydrogen, b) C₂~ C₆Alkenyl, R¹⁰O-, R¹¹S (O)_m-, R¹⁰C (
O) NR¹⁰-, CN, N₃, (R¹⁰)₂NC (NR¹⁰)-, R¹⁰C (
O)-, -N (R¹⁰)₂Or R¹¹OC (O) NR¹⁰Replaced by
Or unsubstituted C₁~ C₆Alkyl, c) unsubstituted or substituted aryl, unsubstituted or substituted heterocycle, unsubstituted or
Substituted cycloalkyl, alkenyl, R¹⁰O-, R¹¹S (O)_m-, R ¹⁰ C (O) NR¹⁰-, CN, NO₂, (R¹⁰)₂NC (NR¹⁰)-,
R¹⁰C (O)-, R¹⁰OC (O)-, N₃, -N (R¹⁰)₂, Halogen
Or R¹¹OC (O) NR¹⁰-And d) aryl, heterocycle and C₃~ C₁₀Selected from cycloalkyl
Unsubstituted or substituted C₁~ C₆Alkyl; R⁴Is

[0105]

Embedded imageR;⁵Represents hydrogen; R⁸Is selected from a) to c) below: a) hydrogen b) C₁~ C₆Alkyl, C₂~ C₆Alkenyl, C₂~ C₆Alkynyl,
C₁~ C₆Perfluoroalkyl, F, Cl, R¹⁰O-, R¹⁰C (O) NR ¹⁰ -, CN, NO₂, (R¹⁰)₂NC (NR¹⁰)-, R¹⁰C (O)-
, R¹⁰OC (O)-, -N (R¹⁰)₂Or R¹¹OC (O) NR¹⁰−
And c) C₁~ C₆Perfluoroalkyl, R¹⁰O-, R¹⁰C (O) NR^{1 0} −, (R¹⁰)₂NC (NR¹⁰)-, R¹⁰C (O)-, R¹⁰OC (O
)-, -N (R¹⁰)₂Or R¹¹OC (O) NR¹⁰C substituted with-₁ ~ C₆Alkyl; R^9aIs independently C₁~ C₆Selected from alkyl and aryl; R¹⁰Is independently hydrogen, C₁~ C₆Alkyl, C₁~ C₆Perfluoroa
Alkyl, 2,2,2-trifluoroethyl, benzyl and aryl
R; R¹¹Is independently C₁~ C₆Choose from alkyl, benzyl and aryl
A;¹And A²Are independently a bond, -CH = CH-, -C≡C-, -C (O
)-, -C (O) NR⁸-, -NR⁸C (O)-, -O-, -N (R⁸)-,-
S (O)₂N (R⁸)-, -N (R⁸) S (O)₂-Or S (O)_mChoose from
V is selected from a) to e) below: a) hydrogen, b) pyrrolidinyl, imidazolyl, pyridinyl, thiazolyl, pyridonyl,
2-oxopiperidinyl, indolyl, quinolinyl, isoquinolinyl and thie
C) aryl, d) 0-4 carbon atoms are replaced by heteroatoms selected from O, S and N.
C₁~ C₂₀Alkyl, and e) C₂~ C₂₀Alkenyl, where A¹Is S (O)_mV is not hydrogen, but A¹V is hydrogen when is a bond
But n is 0, and A²Is S (O)_mZ is H₂Or represents O; m represents 0, 1 or 2; n represents 0, 1, 2, 3 or 4; p independently represents 0, 1, 2, 3 or 4; Represents 0-5, provided that when V is hydrogen, r represents 0; or a pharmaceutically acceptable salt thereof.

In a further embodiment of the compound of formula I according to the present invention, the inhibitor of farnesyl protein transferase is of the formula Ia:

[0107]

Embedded imageWhere R^1bIs independently selected from a) to c) below: a) hydrogen b) aryl, heterocycle, cycloalkyl, R¹⁰O-, -N (R¹⁰)₂ Or C₂~ C₆Alkenyl, c) aryl, heterocycle, cycloalkyl, alkenyl, R¹⁰O- or
−N (R¹⁰)₂C substituted or unsubstituted by₁~ C₆Alkyl
R²Is H; unsubstituted or substituted aryl, or unbranched or branched and unsubstituted
Or C substituted with one or more of the following 1) to 4)_1-5From alkyl
Selected: 1) aryl 2) heterocycle 3) OR⁶Or 4) SR^6aR⁶And R⁷Is independently substituted or substituted with the following a) to d)
Not C_1-4Selected from alkyl, aryl, heteroaryl: a) C_1-4Alkoxy, b) halogen, c) perfluoro-C_1-4Alkyl, or d) aryl or heterocycle; R^6aIs a C substituted or unsubstituted in a) and b) below._1-4Al
Selected from kills: a) C_1-4Alkoxy, or c) aryl or heterocycle; R⁸Is independently selected from a) to c) below: a) hydrogen, b) C_1-6Alkyl, C_2-6Alkenyl, C_2-6Alkynyl, C_{1 to 6} Perfluoroalkyl, F, Cl, R¹⁰O-, R¹⁰C (O) NR¹⁰−,
CN, NO₂, (R¹⁰)₂NC (NR¹⁰)-, R¹⁰C (O)-, -N (
R¹⁰)₂Or R¹¹OC (O) NR¹⁰-And c) C_1-6Perfluoroalkyl, R¹⁰O-, R¹⁰C (O) NR¹⁰ −, (R¹⁰)₂NC (NR¹⁰)-, R¹⁰C (O)-, -N (R¹⁰)₂ Or R¹¹OC (O) NR¹⁰C substituted by-_1-6Alkyl; R¹⁰Is independently hydrogen, C_1-6Alkyl, C_1-6Perfluoroalkyl
, 2,2,2-trifluoroethyl, benzyl and aryl; R¹¹Is independently C_1-6X is -CH₂Z represents a non-alkyl selected from aryl, arylmethyl and arylsulfonyl.
A substituted or substituted group, wherein the substituted group is one of the following a) to k)
Or more than one type: a) C_1-4Alkoxy, NR⁶R⁷, C_3-6Cycloalkyl, unsubstituted
Or substituted aryl, heterocycle, HO, -S (O)_mR^6aOr -C (O) NR ⁶ R⁷Substituted or unsubstituted C_1-4Alkyl, b) aryl or heterocycle, c) halogen, d) OR⁶, E) NR⁶R⁷, F) CN, g) NO₂H) CF₃, I) -S (O)_mR^6a, J) -C (O) NR⁶R⁷Or k) C₃~ C₆M represents 0, 1 or 2; p represents 0, 1, 2, 3 or 4; and r represents 0 to 3; or a pharmaceutically acceptable salt thereof. .

In a further embodiment of the invention, the inhibitor of farnesyl protein transferase is represented by the following formula II-a:

[0109]

Embedded imageWhere R^1bIs independently selected from a) to c) below: a) hydrogen b) aryl, heterocycle, cycloalkyl, R¹⁰O-, -N (R¹⁰)₂ Or C₂~ C₆Alkenyl, c) unsubstituted or substituted aryl, heterocycle, cycloalkyl, alkenyl,
R¹⁰O- or -N (R¹⁰)₂C substituted or unsubstituted by ₁ ~ C₆Alkyl; R^1cIs selected from a) to c) below: a) hydrogen, b) unsubstituted or substituted C₁~ C₆Alkyl, where substituted C₁~ C₆Alkyl
The above substituents are unsubstituted or substituted aryl, heterocycle, C₃~ C₁₀Cycloal
Kill, C₂~ C₆Alkenyl, C₂~ C₆Alkynyl, R¹⁰O-, R¹¹S (
O)_m-, R¹⁰C (O) NR¹⁰−, (R¹⁰)₂NC (O)-, CN, (
R¹⁰)₂NC (NR¹⁰)-, R¹⁰C (O)-, R¹⁰OC (O)-, N ₃ , -N (R¹⁰)₂, And R¹¹OC (O) NR¹⁰-And
C) unsubstituted or substituted aryl;⁶, R⁷And R^7aIs independently H; substituted with the following a) to c)
Is unsubstituted C_1-4Alkyl, C_3-6Cycloalkyl, aryl, f
Selected from terrorist rings: a) C_1-4Alkoxy, b) halogen, or c) aryl or heterocycle; R^6aIs a C substituted or unsubstituted in a) to c) below._1-4Al
Kill or C_3-6Selected from cycloalkyl: a) C_1-4Alkoxy, b) halogen, or c) aryl or heterocycle; R⁸Is independently selected from a) to c) below: a) hydrogen, b) C_1-6Alkyl, C_2-6Alkenyl, C_2-6Alkynyl, C_{1 to 6} Perfluoroalkyl, F, Cl, R¹⁰O-, R¹⁰C (O) NR¹⁰−,
CN, NO₂, (R¹⁰)₂NC (NR¹⁰)-, R¹⁰C (O)-, -N (
R¹⁰)₂Or R¹¹OC (O) NR¹⁰-And c) C_1-6Perfluoroalkyl, R¹⁰O-, R¹⁰C (O) NR¹⁰ −, (R¹⁰)₂NC (NR¹⁰)-, R¹⁰C (O)-, -N (R¹⁰)₂ Or R¹¹OC (O) NR¹⁰C substituted by-_1-6Alkyl; R¹⁰Is independently hydrogen, C_1-6Alkyl, C_1-6Perfluoroalkyl
R, 2,2,2-trifluoroethyl, benzyl and aryl; R¹¹Is independently C_1-6Selected from alkyl and substituted or unsubstituted aryl
Selected; R¹²Is H; unsubstituted or substituted C_1-8Alkyl, unsubstituted or substituted aryl
Selected from unsubstituted or substituted heterocycles, where substituted alkyl, substituted
The reel or substituted hetero ring is substituted with one or more of the following 1) to 10).
1) aryl or unsubstituted aryl or
Is a heterocycle, a) C_1-4Alkyl, b) halogen, c) CN, d) perfluoro-C_1-4Alkyl, 2) C_3-6Cycloalkyl, 3) OR⁶4) SR^6a, S (O) R^6aOr SO₂ ^a,

[0110]

Embedded image 7) N₃, 8) F, 9) Perfluoro-C_1-4-Alkyl, or 10) C_1-6-Alkyl; X¹Is a bond, -C (= O)-or S (= O)_mY is selected from the following a) to c): a) hydrogen b) R¹⁰O-, R¹¹S (O)_m-, R¹⁰C (O) NR¹⁰−, (R^{1 0} )₂NC (O)-, CN, NO₂, (R¹⁰)₂NC (NR¹⁰)-, R ¹² C (O)-, R¹⁰OC (O)-, N₃, F, -N (R¹⁰)₂Or R ¹¹ OC (O) NR¹⁰-, C) unsubstituted or substituted C_1-6Alkyl, where substituted C_1-6On alkyl
The substituent is unsubstituted or substituted aryl, R¹⁰O-, R¹⁰C (O) NR¹⁰−,
(R¹⁰)₂NC (O)-, R¹⁰C (O)-and R¹⁰From OC (O)-
Z is an unsubstituted or substituted aryl, wherein the substituted aryl is any of the following 1) to 11)
Substituted with one or more kinds: 1) C substituted or unsubstituted with the following a) to g)_1-4Al
Kill, a) C_1-4Alkoxy, b) NR⁶R⁷C) C_3-6Cycloalkyl, d) aryl, substituted aryl or heterocycle, e) HO, f) -S (O)_mR^6aOr g) -C (O) NR⁶R⁷, 2) aryl or heterocycle, 3) halogen, 4) OR⁶, 5) NR⁶R⁷, 6) CN, 7) NO₂8) CF₃, 9) -S (O)_mR^6a, 10) -C (O) NR⁶R⁷Or 11) C₃~ C₆M represents 0, 1 or 2; p represents 0 or 2; r represents 0 to 3; and v represents 0, 1 or 2; The salt that can be.

In a further embodiment of the invention, the inhibitor of farnesyl protein transferase is represented by formula III-a:

[0112]

Embedded imageWhere R²Is independently selected from a) to c) below: a) hydrogen b) aryl, heterocycle, cycloalkyl, R¹⁰O-, N (R¹⁰)₂Ma
Or C₂~ C₆Alkenyl, c) aryl, heterocycle, cycloalkyl, alkenyl, R¹⁰O- and again
Is -N (R¹⁰)₂Substituted or unsubstituted C₁~ C₆Alkyl;
 R³Is selected from the following a) to d): a) hydrogen, b) C₂~ C₆Alkenyl, R¹⁰O-, R¹¹S (O)_m-, R¹⁰C (
O) NR¹⁰-, CN, N₃, (R¹⁰)₂NC (NR¹⁰)-, R¹⁰C (
O)-, -N (R¹⁰)₂Or R¹¹OC (O) NR¹⁰Replaced by
Or unsubstituted C₁~ C₆Alkyl, c) substituted or unsubstituted aryl, substituted or unsubstituted heterocycle, C₃~ C₁₀ Cycloalkyl, C₂~ C₆Alkenyl, fluoro, chloro, R¹²O-, R^{1 1} S (O)_m-, R¹⁰C (O) NR¹⁰-, CN, NO₂, (R¹⁰)₂N-
C (NR¹⁰)-, R¹⁰C (O)-, N₃, -N (R¹⁰)₂Or R¹¹ OC (O) NR¹⁰-And d) substituted or unsubstituted aryl, substituted or unsubstituted heterocycle and C₃~ C ₁₀ C substituted with an unsubstituted or substituted group selected from cycloalkyl₁ ~ C₆Alkyl; R⁴And R⁵Is independently selected from a) to d) below: a) hydrogen, b) R¹⁰O- or -N (R¹⁰)₂Replaced or replaced by
Not C₁~ C₆Alkyl, c) substituted or unsubstituted aryl, substituted or unsubstituted heterocycle, C₃~ C₁₀ Cycloalkyl, C₂~ C₆Alkenyl, fluoro, chloro, R¹⁰O-, R^{1 1} S (O)_m-, R¹⁰C (O) NR¹⁰-, CN, NO₂, (R¹⁰)₂N-
C (NR¹⁰)-, R¹⁰C (O)-, N₃, -N (R¹⁰)₂Or R¹¹ OC (O) NR¹⁰-And d) substituted or unsubstituted aryl, substituted or unsubstituted heterocycle and C₃~ C ₁₀ C substituted with an unsubstituted or substituted group selected from cycloalkyl₁ ~ C₆Alkyl; R⁶Is independently selected from a) to c) below: a) hydrogen b) substituted or unsubstituted aryl, substituted or unsubstituted heterocycle, C_1-6Al
Kill, C_2-6Alkenyl, C_2-6Alkynyl, C_1-6Perfluoroalkyl
Le, F, Cl, R¹⁰O-, allyloxy, R¹⁰C (O) NR¹⁰-, CN, N
O₂, (R¹⁰)₂NC (NR¹⁰)-, R¹⁰C (O)-, -N (R¹⁰) ₂ , (R¹²)₂NC (O)-or R¹¹OC (O) NR¹⁰-And c) C_1-6Perfluoroalkyl, R¹⁰O-, R¹⁰C (O) NR¹⁰ −, (R¹⁰)₂NC (NR¹⁰)-, R¹⁰C (O), -N (R¹⁰)₂,
Or R¹¹OC (O) NR¹⁰C substituted with-₁~ C₆Alkyl; R⁷Is independently selected from a) to e) below: a) hydrogen, b) unsubstituted or substituted aryl, c) unsubstituted or substituted heterocycle, d) unsubstituted or substituted cycloalkyl, and e) hydrogen Or selected from aryl, heterocycle and cycloalkyl
C substituted with unsubstituted or substituted groups_1-6Alkyl; wherein the heterocycle is
Pyrrolidinyl, imidazolyl, pyridinyl, thiazolyl, pyridonyl, indori
R, quinolinyl, isoquinolinyl, and thienyl; R⁸Is selected from a) to c) below: a) hydrogen, b) C_1-6Alkyl, C_2-6Alkenyl, C_2-6Alkynyl, C_{1 to 6} Perfluoroalkyl, F, Cl, R¹⁰O-, R¹⁰C (O) NR¹⁰−,
CN, NO₂, (R¹⁰)₂NC (NR¹⁰)-, R¹⁰C (O)-, -N (
R¹⁰)₂Or R¹¹OC (O) NR¹⁰-And c) C_1-6Perfluoroalkyl, R¹⁰O-, R¹⁰C (O) NR¹⁰ −, (R¹⁰)₂NC (NR¹⁰)-, R¹⁰C (O)-, -N (R¹⁰)₂ Or R¹¹OC (O) NR¹⁰C substituted by-_1-6Alkyl; R¹⁰Is independently hydrogen, C_1-6Alkyl, C_1-6Perfluoroalkyl
R, 2,2,2-trifluoroethyl, benzyl and aryl; R¹¹Is independently C_1-6Selected from alkyl and aryl; R¹²Is independently hydrogen, C₁~ C₆Alkyl, CO₂R¹⁰Replaced by
C₁~ C₆C substituted with alkyl or aryl₁~ C₆Alkyl, substituted aryl
C substituted with₁~ C₆Alkyl, heterocyclic substituted C₁~ C₆Alkyl,
C substituted with a substituted heterocycle₁~ C₆Alkyl, aryl and substituted aryl
Selected from: A³Is a bond, -C (O) NR⁷-, -NR⁷C (O)-, -S (O)₂NR ⁷ -, -NR⁷S (O)₂-Or -N (R⁷Z is independently H₂Or represents O; m represents 0, 1 or 2; n represents 0, 1, 2, 3 or 4; p represents 0, 1, 2, 3 or 4; And r represents 0-3; or a pharmaceutically acceptable salt thereof.

In a further embodiment of the compounds of Formula A according to the present invention, the inhibitors of farnesyl protein transferase are of Formula Ai:

[0114]

Embedded imageWhere R^1bIs independently selected from a) to c) below: a) hydrogen b) aryl, heterocycle, cycloalkyl, R¹⁰O-, -N (R¹⁰)₂ Or C₂~ C₆Alkenyl, c) aryl, heterocycle, cycloalkyl, alkenyl, R¹⁰O- or
−N (R¹⁰)₂C substituted or unsubstituted by₁~ C₆Alkyl
R^2aAnd R^2bIs independently selected from a) to d) below: a) hydrogen, b) C₂~ C₆Alkenyl, R¹⁰O-, R¹¹S (O)_m-, R¹⁰C (
O) NR¹⁰-, CN, N₃, (R¹⁰)₂NC (NR¹⁰)-, R¹⁰C (
O)-, R¹⁰OC (O)-, -N (R¹⁰)₂Or R¹¹OC (O) NR ¹⁰ C substituted or unsubstituted by-₁~ C₆Alkyl, c) unsubstituted or substituted aryl, unsubstituted or substituted heterocycle, unsubstituted or
Substituted cycloalkyl, alkenyl, R¹²O-, R¹¹S (O)_m-, R ¹⁰ C (O) NR¹⁰-, CN, NO₂, (R¹⁰)₂NC (NR¹⁰)-,
R¹⁰C (O)-, R¹⁰OC (O)-, N₃, -N (R¹⁰)₂, Halogen
Or R¹¹OC (O) NR¹⁰-And d) aryl, heterocycle and C₃~ C₁₀Selected from cycloalkyl
Unsubstituted or substituted C₁~ C₆Alkyl; R⁴Is

[0115]

Embedded imageR⁵Represents hydrogen; R⁸Is independently selected from a) to c) below: a) hydrogen b) C₁~ C₆Alkyl, C₂~ C₆Alkenyl, C₂~ C₆Alkynyl,
C₁~ C₆Perfluoroalkyl, F, Cl, R¹⁰O-, R¹⁰C (O) NR ¹⁰ -, CN, NO₂, (R¹⁰)₂NC (NR¹⁰)-, R¹⁰C (O)-
, -N (R¹⁰)₂Or R¹¹OC (O) NR¹⁰-And c) C₁~ C₆Perfluoroalkyl, R¹⁰O-, R¹⁰C (O) NR^{1 0} −, (R¹⁰)₂NC (NR¹⁰)-, R¹⁰C (O)-, -N (R¹⁰) ₂ Or R¹¹OC (O) NR¹⁰C substituted with-₁~ C₆Alkyl; R¹⁰Is independently hydrogen, C₁~ C₆Alkyl, substituted or unsubstituted C₁~ C ₆ R is selected from aralkyl, and substituted or unsubstituted aryl;¹¹Is independently C₁~ C₆Choose from alkyl, benzyl and aryl
Z is H₂M represents 0, 1 or 2; n represents 0, 1, 2, 3 or 4; p independently represents 0, 1 or 2; and r represents 0 Or a pharmaceutically acceptable salt thereof.

Particular compounds that are inhibitors of prenyl protein transferase and are therefore useful in the present invention include: 1- [2 (R) -amino-3-mercaptopropyl] -2 (S)- [(3-pyridyl) methoxyethyl)]-4- (1-naphthoyl) piperazine 1- [2 (R) -amino-3-mercaptopropyl] -2 (S)-(benzyloxymethyl) -4- (1 -Naphthoyl) piperazine 1- [2 (R) -amino-3-mercaptopropyl] -2 (S)-(benzyloxymethyl) -4- [7- (2,3-dihydrobenzofuroyl)] piperazine 1- [2 (R) -amino-3-mercaptopropyl] -2 (S)-(benzamido) -4- (1-naphthoyl) piperazine 1- [2 (R) -amino-3-mercaptopropyl Le] -2 (S) - [4- (5
-Dimethylamino-1-naphthalenesulfonamido) -1-butyl] -4- (1
-Naphthoyl) piperazine N- [2 (S)-(1- (4-nitrophenylmethyl) -1H-imidazol-5-ylacetyl) amino-3 (S) -methylphenyl] -N-1-naphthylmethyl-glycyl -Methionine N- [2 (S)-(1- (4-nitrophenylmethyl) -1H-imidazol-5-ylacetyl) amino-3 (S) -methylphenyl] -N-1-naphthylmethyl-glycyl-methionine Methyl ester N- [2 (S)-[(1- (4-cyanobenzyl) -1H-imidazole-5
-Yl] acetylamino) -3 (S) -methylphenyl] -N- (1-naphthylmethyl) -glycyl-methionine N- [2 (S)-[(1- (4-cyanobenzyl) -1H-imidazole -5
-Yl] acetylamino) -3 (S) -methylphenyl] -N- (1-naphthylmethyl) -glycyl-methionine methyl ester 2 (S) -n-butyl-4- (1-naphthoyl) -1- [ 1- (2-naphthylmethyl) imidazol-5-ylmethyl] -piperazine 2 (S) -n-butyl-1-[(1- (4-cyanobenzyl) imidazole-
5-ylmethyl] -4- (1-naphthoyl) piperazine 1-{[1- (4-cyanobenzyl) -1H-imidazol-5-yl] acetyl} -2 (S) -n-butyl-4- (1 -Naphthoyl) piperazine 1- (3-chlorophenyl) -4- [1- (4-cyanobenzyl) imidazolylmethyl] -2-piperazinone 1-phenyl-4- [1- (4-cyanobenzyl) -1H-imidazole-
5-ylethyl] -piperazin-2-one 1- (3-trifluoromethylphenyl) -4- [1- (4-cyanobenzyl) -1H-imidazol-5-ylmethyl] -piperazinone-2-one 1- (3 -Bromophenyl) -4- [1- (4-cyanobenzyl) -1H-imidazol-5-ylmethyl] -piperazinone-2-one 5 (S) -2- [2,2,2-trifluoroethoxy] ethyl) -1- (3-
Trifluoromethylphenyl) -4- [1- (4-cyanobenzyl) -4-imidazolylmethyl] -piperazin-2-one 1- (5,6,7,8-tetrahydronaphthyl) -4- [1- ( 4-cyanobenzyl) -1H-imidazol-5-ylmethyl] -piperazin-2-one 1- (2-methyl-3-chlorophenyl) -4- [1- (4-cyanobenzyl) -4-imidazolylmethyl]- Piperazin-2-one 2 (RS)-{[1- (naphth-2-ylmethyl) -1H-imidazole-5
-Yl) acetyl {amino-3- (t-butoxycarbonyl) amino-N- (2
-Methylbenzyl) propionamide N- {1- (4-cyanobenzyl) -1H-imidazol-5-ylmethyl}
-4 (R) -benzyloxy-2 (S)-{N'-acetyl-N'-3-chlorobenzyl} aminomethylpyrrolidine N- {1- (4-cyanobenzyl) -1H-imidazol-5-ylethyl}
-4 (R) -benzyloxy-2 (S)-{N'-acetyl-N'-3-chlorobenzyl} aminomethylpyrrolidine 1- [1- (4-cyanobenzyl) -1H-imidazol-5-ylacetyl} Pyrrolidin-2 (S) -ylmethyl]-(N-2-methylbenzyl) -glycine N '-(3-chlorophenylmethyl) amide 1- [1- (4-cyanobenzyl) -1H-imidazol-5-ylacetyl} pyrrolidine- 2 (S) -ylmethyl]-(N-2-methylbenzyl) -glycine N'-methyl-N '-(3-chlorophenylmethyl) amide (S) -2-[(1- (4-cyanobenzyl)- 5-imidazolylmethyl) amino] -N- (benzyloxycarbonyl) -N- (3-chlorobenzyl) -4-
(Methanesulfonyl) butanamine 1- (3,5-dichlorobenzenesulfonyl) -3 (S)-[N- (1- (4
-Cyanobenzyl) -1H-imidazol-5-ylmethyl) carbamoyl] piperidine N-{[1- (4-cyanobenzyl) -1H-imidazol-5-yl] methyl} -4- (3-methylphenyl) -4 -Hydroxypiperidine N-{[1- (4-cyanobenzyl) -1H-imidazol-5-yl] methyl} -4- (3-chlorophenyl) -4-hydroxypiperidine 4- [1- (4-cyanobenzyl) -5-imidazolylmethyl] -1- (2,
3-dimethylphenyl) -piperazine-2,3-dione 1- (2- (3-trifluoromethoxyphenyl) -pyrid-5-ylmethyl) -5- (4-cyanobenzyl) imidazole 4- {5- [1 -(3-chloro-phenyl) -2-oxo-1,2-dihydro-pyridin-4-ylmethyl] -imidazol-1-ylmethyl} -2-methoxy-benzonitrile 3 (R) -3- [1- ( 4-cyanobenzyl) imidazol-5-yl-ethylamino] -5-phenyl-1- (2,2,2-trifluoroethyl) -H-benzo [e] [1,4] diazepine 3 (S)- 3- [1- (4-cyanobenzyl) imidazol-5-yl-ethylamino] -5-phenyl-1- (2,2,2-trifluoroethyl) -H-benzo [e] [1,4] Diazepi N- [1- (4-cyanobenzyl) -1H-imidazol-5-ylacetyl) pyrrolidin-2 (S) -ylmethyl] -N- (1-naphthylmethyl) glycyl-methionine N- [1- (4- Cyanobenzyl) -1H-imidazol-5-ylacetyl) pyrrolidin-2 (S) -ylmethyl] -N- (1-naphthylmethyl) glycyl-methionine methyl ester N- [1- (1H-imidazol-4-ylpropionyl) Pyrrolidine-2 (S
) -Ylmethyl] -N- (2-methoxybenzyl) glycyl-methionine N- [1- (1H-imidazol-4-ylpropionyl) pyrrolidine-2 (
S) -ylmethyl] -N- (2-methoxybenzyl) glycyl-methionine methyl ester 2 (S)-(4-acetamido-1-butyl) -1- [2 (R) -amino-3
-Mercaptopropyl] -4- (1-naphthoyl) piperazine 2 (RS)-{[1- (naphth-2-ylmethyl) -1H-imidazole-5
-Yl)] acetyl} amino-3- (t-butoxycarbonyl) amino-N-cyclohexyl-propionamide 1- {2 (RS)-[1- (4-cyanobenzyl) -1H-imidazole-5
-Yl)] propanol {-2 (S) -n-butyl-4- (1-naphthoyl) piperazine 1- [1- (4-cyanobenzyl) imidazol-5-ylmethyl] -4- (
Diphenylmethyl) piperazine 1- (diphenylmethyl) -3 (S)-[N- (1- (4-cyanobenzyl)
-2-Methyl-1H-imidazol-5-ylmethyl) -N- (acetyl) aminomethyl] piperidine N- [1- (1H-Imidazol-4-ylpropionyl) pyrrolidin-2 (S) -ylmethyl] -N- ( 2-chlorobenzyl) glycyl-methionine N- [1- (1H-imidazol-4-ylpropionyl) pyrrolidin-2 (S) -ylmethyl] -N- (2-chlorobenzyl) glycyl-methionine methyl ester 3 (R) -3- [1- (4-Cyanobenzyl) imidazol-5-yl-methylamino] -5-phenyl-1- (2,2,2-trifluoroethyl) -H-benzo [e] [1,4] Diazepine 1- (3-trifluoromethoxyphenyl) -4- [1- (4-cyanobenzyl) imidazolylmethyl] -2-piperazinone 1 (2,5-dimethylphenyl) -4- [1- (4-cyanobenzyl) imidazolylmethyl] -2-piperazinone 1- (3-methylphenyl) -4- [1- (4-cyanobenzyl) imidazolylmethyl] -2-piperazinone 1- (3-iodophenyl) -4- [1- (4-cyanobenzyl) imidazolylmethyl] -2-piperazinone 1- (3-chlorophenyl) -4- [1- (4-cyano-3) -Methoxybenzyl) imidazolylmethyl] -2-piperazinone 1- (3-trifluoromethoxyphenyl) -4- [1- (4-cyano-3-
Methoxybenzyl) imidazolylmethyl] -2-piperazinone 4-[((1- (4-cyanobenzyl) -5-imidazolyl) methyl) amino] benzophenone 1- (1-{[3- (4-cyanobenzyl) -3H] -Imidazol-4-yl
] -Acetyl} -pyrrolidin-2 (S) -ylmethyl) -3 (S) -ethyl-pyrrolidine-2 (S) -carboxylic acid 3-chloro-benzylamide or a pharmaceutically acceptable salt thereof.

As described above as inhibitors of farnesyl protein transferase,
In addition, compounds within the scope of the present invention identified by this assay as inhibitors of farnesyl protein transferase are useful in the present invention, i.e., the methods for their synthesis are described in the following patents, which are incorporated herein by reference: Can be found in applications and publications.

[0118] US Patent No. 5,736,539 (April 7, 1998); WO 95/00
497 (Jan. 5, 1995) US Pat. 5, 652, 257 (July 29, 1997); WO 96/1
0034 (April 4, 1996); WO 96/30343 (October 3, 1996); USSN 08 / 412,829 filed on March 29, 1995; and USSN 08 / 470,690 filed on June 6, 1995. And 1996
US Ser. No. 08 / 600,728, filed Feb. 28, 1998; U.S. Patent No. 5,661,161 (August 26, 1997); 5,756,528 (May 6, 1998); WO 96/39.
137 (dated December 12, 1996); WO 96/37204 (dated November 28, 1996); USSN 08 / 449,038 filed May 24, 1995; USSN filed May 15, 1996
WO 97/18813 (May 29, 1997); USSN 08 / 749,254 filed on November 15, 1996; WO 97/38665 (Oct 23, 1997); April 1, 1997. WO 97/36889 (October 9, 1997); USSN 08 / 823,923, filed March 25, 1997; WO 97/36901 (October 9, 1997); March 1997; WO 97/36879 (October 9, 1997); US Ser. No. 08 / 823,920, filed on March 25, 1997; WO 97/36605 (October 9, 1997); USSN 08 / 823,934 filed March 25; W O98 / 28980 (July 9, 1998); USSN 08 / 997,171 filed on December 22, 1997; and USSN 60 / 014,791 filed on April 3, 1996; April 4, 1997.
USSN 08 / 831,308, dated application.

All patents, publications and pending patent applications identified are hereby incorporated by reference.

For the compounds of the formulas IIa to IIn, the following definitions apply.

The term “alkyl” means a monovalent alkane (hydrocarbon) derived group containing from 1 to 15 carbon atoms unless otherwise specified. It may be linear, branched or cyclic. Preferred straight or branched alkyl groups include methyl, ethyl, propyl, isopropyl, butyl and t-butyl. Preferred cycloalkyl groups include cyclopentyl and cyclohexyl.

When substituted alkyl is present, this means a straight-chain, branched or cyclic alkyl group as defined above, which is substituted with one to three groups as defined for each.

Heteroalkyl means an alkyl group having 2 to 15 carbon atoms and interrupted by 1 to 4 heteroatoms selected from O, S and N.

The term “alkenyl” refers to a straight, branched or cyclic hydrocarbon group containing 2 to 15 carbon atoms and at least one carbon-carbon double bond. Preferably, there is one carbon-carbon double bond and up to four non-aromatic (non-resonant) carbon-carbon double bonds may be present. Examples of alkenyl groups are vinyl, allyl, iso-propenyl, pentenyl, hexenyl, heptenyl, cyclopropenyl, cyclobutenyl,
Including cyclopentenyl, cyclohexenyl, 1-propenyl, 2-butenyl, 2-methyl-2-butenyl, isoprenyl, farnesyl, geranyl, geranylgeranyl and the like. Preferred alkenyl groups include ethenyl, propenyl, butenyl and cyclohexenyl. As described above for alkyl, the straight, branched or cyclic portion of the alkenyl group may contain double bonds and may be substituted if a substituted alkenyl group is provided.

The term “alkynyl” means a straight, branched or cyclic hydrocarbon group containing 2 to 15 carbon atoms and at least one carbon-carbon triple bond. Up to three carbon-carbon triple bonds may be present. Preferred alkynyl groups include ethynyl, propynyl and butynyl. As described above for alkyl, the straight, branched or cyclic portion of the alkynyl group may contain triple bonds and may be substituted if a substituted alkynyl group is provided.

Aryl refers to aromatic rings such as phenyl, substituted phenyl and similar groups, as well as fused rings such as naphthyl and the like. That is, an aryl includes at least one ring having at least 6 atoms, has up to 2 such rings, includes up to 10 atoms therein, and has alternating (resonant) two atoms between adjacent carbon atoms. Has a heavy bond. Preferred aryl groups are phenyl and naphthyl. Aryl groups may be similarly substituted as described below. Preferred substituted aryls include phenyl and naphthyl substituted by one or two groups.
For a farnesyltransferase inhibitor, "aryl" includes any stable monocyclic, bicyclic or tricyclic carbocycle in which at least one ring is aromatic and has up to 7 members in each ring. Is intended. Examples of aryl groups include phenyl, naphthyl, anthracenyl, biphenyl, tetrahydronaphthyl, indanyl, phenanthrenyl, and the like.

The term “heteroaryl” refers to at least one heteroatom O, S or N
Wherein the carbon or nitrogen atom is the point of attachment, one additional carbon atom is optionally substituted by a heteroatom selected from O or S, and 1-3 additional carbon atoms are nitrogen heteroatoms Means a monocyclic aromatic hydrocarbon group having 5 or 6 ring atoms or a bicyclic aromatic group having 8 to 10 atoms optionally substituted by

That is, heteroaryl includes aromatic and partially aromatic groups containing one or more heteroatoms. Examples of this type are thiophene, purine, imidazopyridine, pyridine, oxazole, thiazole, oxazine, pyrazole, tetrazole, imidazole, pyridine, pyrimidine, pyrazine and triazine. Examples of partially aromatic groups are tetrahydroimidazo [4,5-c] pyridine, phthalidyl and saccharinyl, as defined below.

With respect to farnesyltransferase inhibitors, the term heterocycle or heterocyclic as used herein refers to a stable 5-7 membered monocyclic, stable 8-11 membered bicyclic or stable 11-15 membered tricyclic. A cyclic heterocycle, which is saturated or unsaturated, consisting of carbon atoms and from 1 to 4 heteroatoms selected from the group consisting of N, O and S; The formula ring includes any bicyclic group fused to a benzene ring. Heterocyclic rings may be attached to any heteroatom or carbon, thereby forming a stable structure. Examples of such heterocyclic rings are azepinyl, benzimidazolyl, benzisoxazolyl, benzofurazanyl, benzopyranyl, benzothiopyranyl, benzofuryl, benzothiazolyl, benzothienyl, benzoxazolyl, chromanyl, cinnolinyl, dihydrobenzofuryl, dihydro-benzofuryl. Benzothienyl, dihydrobenzothiopyranyl, dihydrobenzothio-pyranyl sulfone, furyl, imidazolidinyl, imidazolinyl, imidazolyl, indolinyl, indolyl, isochromanyl, isoindolinyl, isoquinolinyl, isothiazolidinyl, isothiazolyl, isothiazolidinyl, morpholinyl, naphthyridinyl , Oxadiazolyl, 2-oxoazepinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrroli Nyl, piperidyl, piperazinyl, pyridyl, pyridyl N-oxide, pyridonyl, pyrazinyl, pyrazolidinyl, pyrazolyl, pyrimidinyl, pyrrolidinyl, pyrrolyl, quinazolinyl, quinolinyl, quinolinyl N-oxide, quinoxalinyl, tetrahydrofuryl, tetrahydroisoquinolinyl, tetrahydro-quinolinyl , Thiamorpholinyl, thiamorpholinyl sulfoxide, thiazolyl, thiazolinyl, thienofuryl, thienothienyl and thienyl. Preferably, the heterocycle is selected from imidazolyl, 2-oxopyrrolidinyl, piperidyl, pyridyl and pyrrolidinyl.

With respect to farnesyltransferase inhibitors, “substituted aryl”, “substituted
The terms “heterocycle” and “substituted cycloalkyl” are defined as F, Cl, Br, CF ₃ , NH₂, N (C₁~ C₆Alkyl)₂, NO₂, CN, (C₁~ C₆Archi
Le) O-, -OH, (C₁~ C₆Alkyl) S (O)_m−, (C₁~ C₆Archi
C) C (O) NH-, H₂NC (NH)-, (C₁~ C₆Alkyl) C (O)
−, (C₁~ C₆Alkyl) OC (O)-, N₃, (C₁~ C₆Alkyl) OC
(O) NH- and C₁~ C₂₀Select from the group containing alkyl without limitation
It is intended to include cyclic groups substituted with one or two substituents.

In the methods of the present invention, the disclosed amino acids are identified by both conventional three letters and the one letter abbreviations shown below.

Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Asparagine or Aspartic acid Asx B Cysteine Cys C Glutamine Gln Q Glutamate Glu E Glutamine Glutamine Glutamine Glutamine Glutamine Glutamine K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valin ValV

With respect to the term “CAAX”, the letter “A” represents an aliphatic amino acid and is not limited to alanine.

The compounds used in the present method may have asymmetric centers and occur as racemates, racemic mixtures, and individually as diastereomers, and all possible isomers, including optical isomers, may be used according to the present invention. include. Unless otherwise specified, the listed amino acids are understood to have the natural “L” configuration.

When R ² and R ³ combine to form — (CH ₂ ) _u —, a cyclic group is formed. Examples of such cyclic groups are

[0136]

Embedded image Including, but not limited to.

Further, such cyclic groups may optionally include heteroatoms. Examples of such heteroatom-containing cyclic groups are

[0138]

Embedded image Including, but not limited to.

When R ⁶ and R ⁷ , and R ⁷ and R ^7a combine to form — (CH ₂ ) _u —,
A cyclic group is formed. Examples of such cyclic groups are

[0140]

Embedded image Including, but not limited to.

The pharmaceutically acceptable salts of the compounds of the present invention include the conventional non-toxic salts of the compounds of the present invention as formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts are those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, etc .; acetic acid, propionic acid, succinic acid, glycolic acid , Stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, 2-
Includes salts prepared from organic acids such as acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethionic acid, trifluoroacetic acid and the like.

Any substituent or variable at a particular position in the molecule (eg, R ¹⁰ , Z,
n, etc.) is intended to be independent of its definition elsewhere in the molecule. That, ^{-N (R} _{10) 2} represents _-NHH, -NHCH _3, a -NHC ₂ H ₅ and the like. The substituents and substitution patterns on the compounds of the present invention are chemically stable and provide compounds that can be readily synthesized by techniques known in the art as well as the methods described below. It is understood that it can be selected by those skilled in the art.

Pharmaceutically acceptable salts of the compounds of the present invention can be synthesized from the compounds of the present invention that contain a basic group by conventional chemical methods. Usually, the salts are prepared by reacting the free base with a stoichiometric or excess of the desired salt-forming inorganic or organic acid in a suitable solvent or various combination solvents.

The compounds used in the methods of the present invention are useful in various pharmaceutically acceptable salt forms. The term "pharmaceutically acceptable" refers to a salt state apparent to the pharmacist of chemists, ie, substantially non-toxic, having the desired pharmacokinetic properties, palatability, absorbability,
Means that provide dispersibility, metabolism or excretion. Other factors of more practical nature, which are also important in selection, are raw material cost, ease of crystallization, yield, stability,
Hygroscopicity, and the fluidity of the resulting bolus. Conventionally, pharmaceutical compositions can be prepared from the active ingredient in combination with a pharmaceutically acceptable carrier.

The pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts formed, for example, from non-toxic inorganic or organic acids. Non-toxic salts are those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric acids and the like;
Acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid
, Sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid,
Includes salts prepared from organic acids such as methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethionic acid, trifluoroacetic acid and the like.

The pharmaceutically acceptable salts of the present invention can be synthesized by conventional chemical methods. Usually, the salts are prepared by reacting the free base with a stoichiometric or excess of the desired salt-forming inorganic or organic acid or base in a suitable solvent or various combination solvents.

The abbreviations used in the chemical description and examples are shown below:

Ac ₂ O Acetic anhydride; Boc t-butoxycarbonyl; DBU 1,8-diazabicyclo [5.4.0] undec-7-ene; DMAP 4-dimethylaminopyridine; DME 1,2-dimethoxyethane; DMF Dimethylformamide EDC 1- (3-dimethylaminopropyl) -3-ethyl-carbodiimid-hydrochloride HOBT 1-hydroxybenzotriazole hydrate; Et ₃ N triethylamine EtOAc ethyl acetate FAB fast atom bombardment HOOBT 3-hydroxy-1, 2,2-benzotriazin-4 (3H) -one; HPLC high performance liquid chromatography MCPBA m-chloroperoxybenzoic acid MsCl methanesulfonyl chloride NaHMDS sodium bis (trimethylsilyl) amide; Py pyr Gin TFA Trifluoroacetic acid THF Tetrahydrofuran

The prenyltransferase inhibitors of formula I can be used in addition to other standard procedures, such as ester hydrolysis, cleavage of protecting groups, etc. known in the literature or exemplified in experimental procedures, as well as Schemes 1-11. Can be synthesized by The substituents R, R ^a and R ^b shown in the schemes represent the substituents R ² , R ³ , R ⁴ and R ⁵ ; however, the point at which they add to the ring is shown for illustration only and is not intended to be limiting. Absent.

These reactions can be used sequentially and sequentially to provide compounds of the present invention, or they can be used to synthesize fragments that are subsequently coupled by the alkylation reactions described in the Schemes. Can be.

[0151] Schematic of schemes 1-11:  The intermediate sought is, in some cases, commercially available and, in most cases,
It can be prepared by a procedure.

Piperazin-5-one can be prepared as shown in Scheme 1. That is, the protected and appropriately substituted amino acid IV can be converted to the corresponding aldehyde by first forming an amide and then reducing it with LAH. Bo
Reductive amination of c-protected amino aldehyde V gives compound VI. Intermediate VI is acylated with chloroacetyl chloride to give VII,
Conversion to piperazinone can be achieved by base-induced cyclization to III. Deprotection followed by reductive alkylation with a protected imidazole carboxaldehyde gives IX, which can be alkylated with an arylmethyl halide to give the imidazolium salt X. First, the protecting group is removed by solvolysis with a lower alkyl such as methanol, or by treatment with triethylsilane in methylene chloride in the presence of trifluoroacetic acid to give the final product XI.

Intermediate VIII can be reductively alkylated with various aldehydes such as XII. Aldehydes are derived from the appropriate amino acids by O.D. P. Goel
, U.S. Krolls, M .; Stier and S.M. Kesten al, Organi c Syntheses, 1988 years, Vol. 67, can be prepared using standard procedures such as those described in pages 69 to 75 (Scheme 2). Reductive alkylation can be achieved at pH 5-7 using various reducing agents such as sodium triacetoxyborohydride or sodium cyanoborohydride in a solvent such as dichloroethane, methanol or dimethylformamide.
Product XIII can be deprotected with trifluoroacetic acid in methylene chloride to give final compound XIV. The final product XIV is isolated, for example, in the form of a salt such as, for example, trifluoroacetate, hydrochloride or acetate. The product diamine XIV can be further selectively deprotected to give XV, which is subsequently
Subsequent reductive alkylation with an aldehyde can provide XVI. Removal of the protecting group and conversion to a cyclized product such as dihydroimidazole XVII can be accomplished by literature procedures.

Alternatively, imidazole acetate XVIII can be converted to XIX acetate by standard procedures, where XIX is first reacted with an alkyl halide and then treated with methanol at reflux to give a regiospecific alkylation. The imidazole acetate XX can be obtained (Scheme 3). Hydrolysis and reaction with piperazinone VIII in the presence of a condensing reagent such as 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (EDC) gives an acylated product such as XXI.

Reductive alkylation of piperazinone VIII with an aldehyde that also has a protected hydroxy group, such as XXII in Scheme 4, allows the subsequent removal of the protecting group to remove the hydroxyl group (Scheme 4, 5). The alcohol can be oxidized under standard conditions to, for example, an aldehyde, which can then be reacted with various organometallic reagents, such as Grignard reagents, to give a secondary alcohol, such as XXIV. In addition, the fully deprotected amino alcohol XXV can be reductively alkylated (under the conditions described above) with various aldehydes to give a secondary or tertiary amine such as XXVI (Scheme 5). Can be obtained.

Utilizing Boc-protected amino alcohol XXIII, 2-aziridinylmethylpiperazinone such as XXVII (Scheme 6) can be synthesized. XXII
Treatment of I with 1,1′-sulfonyldiimidazole and sodium hydride in a solvent such as dimethylformamide gives aziridines XXVII. Reaction of aziridine in the presence of a nucleophile such as a thiol in the presence of a base gives the ring-opened product XXVIII.

In addition, piperazinone VIII is reacted with an aldehyde derived from an amino acid such as O-alkylated tyrosine according to standard procedures to form XXX (Scheme 7).
) Can be obtained. When R 'is an aryl group, XXX can be first hydrogenated to remove the phenol and the amine group deprotected with an acid to produce XXXI. Also, the amine protecting group in XXX can be removed to produce an O-alkylated phenolamine such as XXXII.

Scheme 8 shows that an optionally substituted homoserine lactone can be converted to Bo Bo using XXXIII.
It illustrates the preparation of c-protected piperazinone. Intermediate XXXVII is
As illustrated in the previous scheme, it can be deprotected and reductively alkylated or acylated. Also, the hydroxyl group of intermediate XXXVII can be mesylated and replaced with a suitable nucleophile, such as the sodium salt of ethanol thiol, to give intermediate XXXVIII. Intermediate XXXVII is oxidized to intermediate I
A carboxylic acid can be provided on the XL and used to form an ester or amide group.

N-aralkyl-piperazin-5-one can be prepared as shown in Scheme 9. Boc-protected amino aldehyde V (prepared from III as described above)
Is reductively aminated to give compound XL. This is followed by Schott
Reaction with bromoacetyl bromide under en-Baumann conditions; ring closure with a base such as sodium hydride in a polar aprotic solvent such as dimethylformamide. The carbamate protecting group is removed under acidic conditions such as trifluoroacetic acid in methylene chloride, or hydrogen chloride in methanol or ethyl acetate, and the resulting piperazine is then purified to the final product as described in Schemes 1-7. Can be carried on objects.

The isomeric piperazin-3-one can be prepared as described in Scheme 10. The imine formed from arylcarboxamide XLII and 2-aminoglycinal diethyl acetal (XLIII) can be reduced under various conditions including sodium triacetoxyborohydride in dichloroethane to give amine XLIV. Amino acid I is conjugated to amine XLIV under standard conditions and the resulting amide XLV is treated with an aqueous acid in tetrahydrofuran to give the unsaturated XLVI
Can be cyclized. Catalytic hydrogenation under standard conditions gives the desired intermediate XLVII, which is processed as described in Schemes 1-7 to give the final product.

Amino acids of general ILs having side chains not found in natural amino acids can be prepared by reacting as described in Scheme 11, starting from the readily prepared imine XLVIII.

[0162]

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[0163]

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[0164]

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[0165]

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[0166]

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[0167]

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[0168]

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[0169]

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[0170]

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[0171]

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[0172]

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[0173]

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[0174]

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[0175]

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[0176]

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[0177]

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The reactions used to produce compounds of formula (II) are subject to other standard procedures, such as ester hydrolysis, cleavage of protecting groups, etc. known in the literature or exemplified in the experimental procedures. In addition, they are prepared by using reactions as shown in Schemes 16-37. The substituents R ^a and R ^b shown in the scheme are the substituents R ² , R ³ ,
Represents R ⁴ and R ⁵ ; “substituted” for a substituent represents a suitable substituent on substituent Z. The point at which such substituents are added to the ring is shown by way of illustration only, and not limitation.

These reactions can be used sequentially and sequentially to provide compounds of the present invention, or they can be used to synthesize fragments that are subsequently coupled by the alkylation reactions described in the Schemes. Can be.

[0180] Schematics of schemes 16-37:  The intermediates sought are in some cases commercially available and can be prepared by literature procedures.
can do. For example, in Scheme 16, the appropriately substituted Boc-protected
Deprotonate sonipecotate LI and then add the appropriately substituted benzyl bromide.
Treatment with such an appropriately substituted alkylating group affords the gem disubstituted intermediate LIII.
Can be Deprotection and reduction lead to hydroxymethylpiperidine LIV
Obtained, which can be utilized for the synthesis of the compounds of the invention, or nitrogen protection
To give the intermediate LV.

As shown in Scheme 17, the protected piperidine intermediate LIII was deprotected and 1-
Reductive alkylation with an aldehyde such as trityl-4-imidazolyl-carboxaldehyde or 1-trityl-4-imidazolyl acetaldehyde to produce LVI
Can be obtained. The trityl protecting group is removed from LVI to remove L
VII can be obtained, or LVI can be treated first with an alkyl halide and then deprotected to give an alkylated imidazole LVIII.

Deprotected intermediate LIII can also be reductively alkylated with various other aldehydes and acids, as shown in Schemes 4-7.

The synthesis of another, hydroxymethyl intermediate LIV and its use in the synthesis of compounds of the present invention that incorporate a preferred imidazolyl group is illustrated in Scheme 18. Scheme 19 illustrates the reductive alkylation of intermediate LIV to give 4-cyanobenzylimidazolyl-substituted piperidine. The cyano group can be selectively hydrolyzed with sodium borate to give the corresponding amide compound of the invention.

Scheme 20 illustrates another preparation of the methyl ether intermediate LV and a method of alkylating LV with an appropriately substituted imidazolylmethyl chloride to provide the compound.
The preparation of the homologous 1- (imidazolylethyl) piperidine is illustrated in Scheme 21.

Specific substitution of compounds of the invention on piperidine can be achieved as shown in Scheme 22. That is, metal-halogen exchange coupling of a butynyl group to isonicotinate and subsequent hydrogenation provides a 2-butylpiperidine intermediate, which is then subjected to the reactions described above to provide compounds of the present invention. Can be provided.

Incorporating a 4-amide group for LV is shown in Scheme 23.

Scheme 24 illustrates the synthesis of compounds of the invention where the Z group was added directly to the piperidine ring. That is, gem disubstituted piperidine LX is obtained by treating piperidone LIX with an appropriately substituted phenyl Grignard reagent. Deprotection gives the key intermediate LXI. The intermediate LXI can be acetylated as described above to give the compound LXII (Scheme 25).

As shown in Scheme 26, the protected piperidine LX can be dehydrated and then borohydride to provide 3-hydroxypiperidine LXIII. This compound is deprotected and further derivatized to provide a compound of the invention (as shown in Scheme 27).
Alternatively, the hydroxy group can be alkylated as shown in Scheme 26 prior to deprotection and further processing.

The dehydrated product is catalytically reduced to provide the des-hydroxy intermediate LXV, as shown in Scheme 28, which can be processed by the reactions described in the previous scheme.

Schemes 29 and 30 illustrate that the 4-carboxylic acid functionality is further chemically treated to provide compounds of the invention wherein the substituent Y is an acetylamine or sulfonamide group.

Scheme 31 illustrates the incorporation of a nitrile group at the 4-position of piperidine in compounds of formula II. That is, the appropriately substituted 4-hydroxypiperidine hydroxyl group is replaced with a nitrile to give the intermediate LXVI, which is depicted in Scheme 1
It can be subjected to the reaction described above in 7-21.

Scheme 32 illustrates the preparation of some pyridyl intermediates that can be utilized with piperidine intermediates such as compound LI of Scheme 16 to obtain compounds of the present invention. Scheme 33 illustrates a general reaction sequence utilizing such a pyridyl intermediate.

Compounds of the present invention wherein X ¹ is a carbonyl group can be prepared as shown in Scheme 34. Intermediate LXVII can be subjected to the following reaction as shown in Schemes 17-21 to provide the compounds of the present invention. The preparation of this compound where X ¹ is sulfur in various oxidation states is shown in Scheme 35. Intermediate LXVIII-LXXI is
Can be subjected to the reactions described above to provide the compounds of the present invention.

Scheme 36 provides the compounds of formula A wherein Y is hydrogen. That is,
The appropriately substituted isonipeconic acid can be treated with N, O-dimethylhydroxylamine and the intermediate LXXII can be reacted with an appropriately substituted phenyl Grignard reagent to give the intermediate LXXIII. This intermediate can be subjected to reactions as previously described in Schemes 17-21 and further modified by reduction of phenyl ketone to give alcohol LXXIV.

Compounds of the present invention wherein X ¹ is an amine group can be prepared as shown in Scheme 37. That is, N-protected 4-piperidinone can be reacted with an appropriately substituted aniline in the presence of trimethylsilyl cyanide to provide 4-cyano-4-aminopiperidine LXXV. Intermediate LXXV can then be converted sequentially to the corresponding amide LXXVI, ester LXXVII, and alcohol LXXVIII. Deprotection of intermediate LXXVI-LXXVIII followed by Scheme 17
To 21 to provide the compounds of the present invention.

[0196]

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[0197]

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[0198]

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[0199]

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[0200]

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[0201]

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[0219]

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The compounds of the invention of formula (III) can be prepared, in addition to other standard procedures such as ester hydrolysis, cleavage of protecting groups and the like, known in the literature or exemplified in the experimental procedures, by It is prepared by using the reaction shown in Reaction Schemes 38-51. Some important bond formation and peptide modification reactions are shown below:

Reaction A : Amide bond formation and protecting group decomposition reaction using standard solution or solid phase methods B : Reduced peptide subunit by reductive alkylation of amine with aldehyde using sodium cyanoborohydride or other reducing agent Unit Preparation Reaction C : Alkylation of Reduced Peptide Subunit with Alkyl or Alkyl Halide, or Reductive Alkylation Reaction of Reduced Peptide Subunit with Aldehyde Using Sodium Cyanoborohydride or Other Reducing Agents D : Peptide bond formation and protecting group degradation using standard solution or solid-phase methods
Reaction E : Preparation of reduced subunit by borane reduction of amide group

These reactions can be used sequentially and sequentially to provide compounds of the present invention, or they can be used by alkylation reactions described in these schemes and in Schemes 43-51 below. Subsequent ligated fragments can be synthesized.

[0223]

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[0224]

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[0228] Here, ^{R A} and ^{R B} and ^R 2, ^{R 3} or ^{R 5} are as defined above; ^{R C} and ^{R D} is an ^{R 7} or ^{R 12; X L} is a leaving group, for example , ^Br -, I ^- or MsO ^- a is; and Ry are defined as ^{R 7} is caused by the reductive alkylation process.

In addition to the reactions described in Schemes 26-30, other reactions used to produce compounds of Formula (III) of the present invention are shown in Schemes 43-51. The substituents shown in these reaction schemes all represent the same substituents as defined above. The substituent "Ar" in the reaction schemes represents a carbocyclic or heterocyclic, substituted or unsubstituted aromatic ring.

These reactions can be used sequentially and sequentially to provide compounds of the present invention, or they can be used to synthesize fragments that are subsequently linked by the alkylation reactions described in these reaction schemes. can do. The sequential order in which the substituents are incorporated into the compounds is often not important, and the order of reactions described in the reaction schemes is illustrative only and not limiting.

Overview of Schemes 43-51 : The required intermediates may be commercially available or may be readily prepared according to known literature procedures, including those described in Schemes 38-42 above. it can.

Reaction Scheme 43 illustrates the incorporation of a cyclic amine group, such as a reduced prolyl group, into compounds of Formula III of the present invention. Reduction of the azide LXXXI gives the amine LXXXII, which can be mono- or disubstituted using the techniques described above. As an example, the incorporation of a naphthylmethyl group and an acetyl group will be described.

As shown in Reaction Scheme 44, direct addition of an aromatic ring to a substituted amine such as LXXXIII is achieved by coupling with a triarylbismuth agent such as tris (3-chlorophenyl) bismuth. You.

Reaction Scheme 45 illustrates the use of protecting groups to prepare compounds of the invention where the cyclic amine contains an alkoxy group. The hydroxy group of the key intermediate LXXXIVa can be further converted to a fluoro or phenoxy group, as shown in Scheme 46. The intermediates LXXXV and LXXXVI can then be further processed to provide the compounds.

[0235] Scheme 474, variables _{^{- (CR 4 2) q A}} 3 (CR 5 2) n R 6 is described the synthesis of the compound which is appropriately substituted α- hydroxybenzyl group. That is, treatment of the protected intermediate aldehyde with an appropriately substituted phenyl Grignard reagent provides the enantiomeric mixture LXXXVII. By treating the mixture with 2-pyrolinyl chloride, compounds LXXXVIII and IXC can be resolved by chromatography. Removal of the pyrrolinoyl group followed by deprotection provides the optically pure intermediate XC, which can be further processed as described above to give the compound.

[0236] shown in variables p is 0 or 1 and Z is the reaction synthesis of useful imidazole-containing intermediates in the synthesis of the compound that is with H ₂ graphically 48 and 49. That is, mesylate XCI can be used to alkylate suitably substituted amines or cyclic amines, while aldehyde XCII can be used to reductively alkylate such amines as well.

[0237] Scheme ^{_{50, - (CR 2 2) p}} -C (Z) - the point of attachment to W (imidazolyl) groups is described the synthesis of imidazole containing intermediate is via an imidazole ring nitrogen. Scheme 51, R ² substituent is described the synthesis of intermediates is methyl.

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The prenyltransferase inhibitors of formula (A) can be used in addition to other standard procedures such as ester hydrolysis, cleavage of protecting groups, etc. known in the literature or exemplified in the experimental procedures, Can be synthesized according to the following reaction scheme. 2 shows some important reactions utilized for the formation of the aminodiphenyl group of the present compounds.

These reactions can be used sequentially and sequentially to provide compounds of the present invention, or they can be used to synthesize fragments that are subsequently linked by the alkylation reactions described in these reaction schemes. can do.

A method for forming the benzophenone intermediate shown in Reaction Scheme 52 is a Stille reaction using an arylstannane. Next, such an amine intermediate is
As explained above, it can be reacted with various aldehydes and esters / acids.

[0253]

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【Example】

The examples provided are intended to aid a further understanding of the invention. The particular materials, species, and conditions used are intended to provide a further description of the invention and are not intended to limit the reasonable scope of the invention.

The standard procedure referred to in the examples is solvent extraction and 10% citric acid, 10%
Washing of the organic solution with sodium bicarbonate and a suitable brine is meant. The solution is dried over sodium sulfate and evaporated under reduced pressure on a rotary evaporator.

Example 1 1- (3-Chlorophenyl) -4- [1- (4-cyanobenzyl) imidazolylmethyl] -2-piperazinone dihydrochloride (Compound 1) Step A: 1-Triphenylmethyl-4- Preparation of (hydroxymethyl) -imidazole 4- (Hydroxymethyl) imidazole hydrochloride (3
5.0 g (260 mmol) in a solution containing trimethylamine (90.
(6 mL, 650 mmol) was added. A white solid precipitated from the solution. DMF
Chlorotriphenylmethane (76.1 g, 273 mmol) in 500 mL was added dropwise. The reaction mixture was stirred for 20 hours, poured on ice, filtered and washed with ice water. The resulting product was slurried with cold dioxane, filtered and dried under reduced pressure to give the title compound as a white solid sufficiently pure for the next step.

Step B: Preparation of 1-triphenylmethyl-4- (acetoxymethyl) -imidazole The alcohol from Step A (260 mmol, previously prepared) was combined with 500 mL of pyridine.
Suspended in it. Acetic anhydride (74 mL, 780 mmol) was added dropwise, and the reaction mixture was diluted with 4 parts.
After stirring for 8 hours, the reaction became homogeneous. The solution is poured into 2 L of EtOA and washed with water (3 × 1 L), 5% aqueous HCl (2 × 1 L), saturated aqueous NaHCO ₃ , and brine, then dried (Na ₂ SO ₄ ), filtered and reduced in vacuo Concentrated down to give the crude product. The acetate salt was isolated as a white powder that was pure enough for the next reaction.

Step C: Preparation of 1- (4-cyanobenzyl) -5- (acetoxymethyl) -imidazole hydrobromide The product from Step B (85.8 g, 225 mmol) in 500 mL of EtOAc
) And α-bromo-p-tolunitrile (50.1 g, 232 mmol) was stirred at 60 ° C. for 20 hours, during which a pale yellow precipitate formed. The reaction was cooled to room temperature and filtered to give a solid imidazolium bromide salt. The filtrate was concentrated under reduced pressure to a volume of 200 mL, reheated at 60 ° C. for 2 hours, cooled to room temperature and filtered again. The filtrate was concentrated under reduced pressure to a volume of 100 mL, reheated at 60 ° C. for a further 2 hours, cooled to room temperature and concentrated under reduced pressure to give a pale yellow solid. All of the solids were combined, dissolved in 500 mL of methanol and warmed to 60 ° C. After 2 hours, the solution was re-concentrated under reduced pressure to give a white solid, which was triturated with hexane to remove soluble material.
The remaining solvent was removed under reduced pressure to give the title product hydrobromide as a white solid, which was used in the next step without further formation.

Step D: Preparation of 1- (4-cyanobenzyl) -5- (hydroxymethyl) -imidazole The acetate from step C (50.4 g, 150 mm) in 1.5 L of 3: 1 THF / water
ol) at 0 ° C. with lithium hydroxide monohydrate (18.9 g, 45
0 mmol) was added. After 1 hour, the reaction was concentrated under reduced pressure and EtOAc (3
L) and washed with water, saturated aqueous NaHCO ₃ and brine. The solution was then dried (Na ₂ SO ₄ ), filtered and concentrated under reduced pressure to give a crude product that was pure enough to be used in the next step without further purification, as a pale yellow cotton-like solid As obtained.

Step E: Preparation of 1- (4-cyanobenzyl) -5-imidazolecarboxaldehyde The alcohol from Step D (21.5 g, 101 mmol) in 500 mL of DMSO
To a solution containing l), was added triethylamine (56 mL, 402 mmol) at room temperature, then SO ₃ - pyridine complex (40.5 g, 254 mmol) was added. After 45 minutes, pour the reaction into 2.5 L of EtOAc, wash with water (4 × 1 L) and brine, dry (Na ₂ SO ₄ ), filter and concentrate under reduced pressure to further purify the aldehyde. But was obtained as a white powder pure enough to be used in the next step.

Step F: Preparation of N- (3-chlorophenyl) ethylenediamine hydrochloride 3-Chloroaniline (30.0 mL, 284 m
mol) at 0 ° C. in 1,4-dioxane (80 mL, 320 mmol).
HCl) in 4N HCl. The solution was warmed to room temperature and then concentrated to dryness under reduced pressure to give a white powder. This powder and 2-oxazolidinone (24.6
g, 282 mmol) under a nitrogen atmosphere at 160 ° C. for 10 hours,
During that time, the solid was melted and gas generation was observed. The reaction was cooled and the crude diamine hydrochloride formed as a light brown solid.

Step G: Preparation of N- (tert-butoxycarbonyl) -N ′-(3-chlorophenyl) ethylenediamine The amine hydrochloride from Step F (about 282 mmol, the crude product previously prepared) was converted to T
Take up in 500 mL of HF and 500 mL of saturated aqueous NaHCO ₃ , cool to 0 ° C., and di-tert-butyl pyrocarbonate (61.6 g, 282 mmol)
Was added. After 30 h, the reaction was poured into EtOAc, washed with water and brine, dried (Na ₂ SO ₄ ), filtered and concentrated under reduced pressure to give the title carbamate as a brown oil, which was purified without further purification. Used in the step.

Step H: N- [2- (tert-butoxycarbamoyl) ethyl] -N- (3
- chlorophenyl) -2 Preparation of chloroacetamide CH ₂ Cl ₂ 500 mL product from Step G in (77 g, approximately 282mmol
) And triethylamine (67 mL, 480 mmol) were cooled to 0 ° C. Chloroacetyl chloride (25.5 mL, 320 mmol) was added dropwise and the reaction was maintained at 0 ° C. with stirring. After 3 hours, additional chloroacetyl chloride (3.0
mL) was added dropwise. After 30 minutes, pour the reaction into EtOAc (2 L) and add water, sat.
Washed with aqueous H ₄ Cl, saturated aqueous NaHCO ₃ and brine. The solution was dried (Na ₂ SO ₄ ), filtered and concentrated under reduced pressure to give chloroacetamide as a brown oil, which was used for the next step without further purification.

Step I: Preparation of 4- (tert-butoxycarbamoyl) -1- (3-chlorophenyl) -2-piperazinone The chloroacetamide from Step H (about 282 mmol) in 700 mL dry DMF
A solution containing _l), was added to _{K 2 CO 3 (88g, 0.64mol} ). The solution was heated in an oil bath at 70-75 ° C. for 20 hours, cooled to room temperature, and concentrated under reduced pressure to remove about 500 mL of DMF. The remaining material was poured into 33% EtOAc / hexane, washed with water and brine, dried (Na ₂ SO ₄ ), filtered and concentrated under reduced pressure to give the product as a brown oil. This material was purified by silica gel chromatography (25-50% EtOAc / hexane) to give a sample of the pure product as well as a product containing less polar impurities (about 65% pure by HPLC).

Step J: Preparation of 1- (3-chlorophenyl) -2-piperazinone Boc-protected piperazinone from Step I (17.1) in 500 mL of EtOAc.
9 g, 55.4 mmol) at −78 ° C. with anhydrous HCl gas.
I do. The saturated solution was warmed to 0 ° C. and stirred for 12 hours. Nitrogen gas is blown into the reaction solution
The excess HCl was removed by heating and the mixture was warmed to room temperature. Concentrate the solution under reduced pressure
The hydrochloride was obtained as a white powder. This material is CH₂Cl₂Take in 300 mL,
Dilute NaHCO₃Treated with aqueous solution. The aqueous phase is removed until tlc analysis indicates complete extraction.
, CH₂Cl₂(8 × 300 mL). Dry the combined organic mixture (Na ₂ SO₄), Filter and concentrate under reduced pressure to give the free amine as a pale brown oil.
Obtained.

Step K: Preparation of 1- (3-chlorophenyl) -4- [1- (4-chlorobenzyl) imidazolylmethyl] -2-piperazinone dihydrochloride The amine from step J in 200 mL of 1,2-dichloroethane (55.4mmo
1, 4 g of powder molecular sieve (10 g) at 0 ° C.
Subsequently, sodium triacetoxyborohydride (17.7 g, 83.3 mmol)
Was added. The imidazole carboxaldehyde from Step E of Example 1 (11.
9 g, 56.4 mmol) was added and the reaction was stirred at 0 ° C. After 26 hours, the reaction was poured into EtOAc, washed with dilute aqueous NaHCO _{3 and the} aqueous layer was back-extracted with EtOAc. The combined organics were washed with brine, dried (Na ₂ SO ₄ ), filtered,
Concentrated under reduced pressure. The obtained product is mixed with 5: 1 benzene: CH ₂ Cl ₂ 500 mL
And propylamine (20 mL) was added. The mixture was stirred for 12 hours and then concentrated under reduced pressure to give a pale yellow oil. This material was purified by silica gel chromatography (2-7% MeOH / CH ₂ Cl ₂ ) and the resulting white foam was taken up in CH ₂ Cl ₂ and treated with 2.1 equivalents of a 1 M HCl / ether solution. . After concentration under reduced pressure, the product dihydrochloride was isolated as a white powder.

The products of Examples 2-5 (Table 1) were combined with the structurally related compounds 1- (3-
Chlorophenyl) -4- [1- (4-cyanobenzyl) imidazolylmethyl]-
Prepared using the above protocol describing the synthesis of 2-piperazinone dihydrochloride. In step F, an appropriately substituted aniline was used instead of 3-chloroaniline.

Table 1: 1-Aryl-4- [1- (4-cyanobenzyl) imidazolylmethyl] -2-piperazinone

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[0269]

[Table 1]

Example 6 1- (3-Chlorophenyl) -4- [1- (4-cyano-3-methoxybenzyl) imidazolylmethyl] -2-piperazinone dihydrochloride Step A; 4-Amino-3-hydroxybenzoic Preparation of methyl acid salt 4-amino-3-hydroxybenzoic acid (75 g,
0.49 mol) was blown at room temperature with anhydrous HCl gas until the solution was saturated. The solution was stirred for 48 hours and then concentrated under reduced pressure. The product is treated with EtOA
c and saturated aqueous NaHCO ₃ , the organic layer was washed with brine, dried (Na ₂ SO ₄ ) and concentrated under reduced pressure to give the title compound (79 g, 96% yield).

Step B: Preparation of Methyl 3-Hydroxy-4-iodobenzoate The product from Step A (79 g, 0.47 mol), 3N HCl (750 mL)
) And THF (250 mL) were cooled to 0 ° C. Water 1
NaNO ₂ (35.9g, 0.52mol) in 15mL was added over the solution for about 5 minutes including the solution was stirred for an additional 25 minutes. Potassium iodide (312 g,
(1.88 mol) in 235 mL of water was added at once, and the reaction was stirred for an additional 15 minutes. The mixture was poured into EtOAc, shaken and the layers were separated. The organic phase was washed with water and brine, dried _(Na 2 SO _4), was obtained and concentrated under reduced pressure the crude product (148 g). Column chromatography through silica gel (0% ~
Purify by 50% EtOAc / hexane to give the title product (96 g, 73% yield)
) Got.

Step C: Preparation of methyl 4-cyano-3-hydroxybenzoate The iodide product from Step B (101 g, 0.36 m) in 400 mL dry DMF
)) and zinc (II) cyanide (30 g, 0.25 mol) were degassed by bubbling argon through the solution for 20 minutes. Tetrakis (triphenylphosphine) palladium (8.5 g, 7.2 mmol) was added and the solution was heated at 80 ° C. for 4 hours. The solution was cooled to room temperature and then stirred for a further 36 hours. The reaction was poured into EtOAc / water and the organic layer was washed with brine (4 ×) and dried (N
a ₂ SO ₄ ) and concentrated under reduced pressure to give the crude product. Purification by column chromatography over silica gel (30% to 50% EtOAc / hexane) provided the title product (48.8 g, 76% yield).

Step D: Preparation of Methyl 4-cyano-3-methoxybenzoate Sodium hydride (9 g, 0.24 mol, 60% by weight mineral oil dispersion) was added at room temperature to the phenol from Step C in 400 mL of dry DMF ( 36.1g, 204m
mol)). Iodomethane (14 mL, 0.22 mol) was added and the reaction was stirred for 2 hours. Pour the mixture into EtOAc / water, wash the organic layer with water and brine (4 ×), dry (Na ₂ SO ₄ ) and concentrate under reduced pressure to give the title product (37.6 g, 96% yield). I got

Step E: Preparation of 4-cyano-3-methoxybenzyl alcohol The ester from Step D (48.8 g, 255 mmol) in 400 mL of dry THF
l) in a solution containing lithium borohydride (255 m
L, 510 mmol, 2M THF) was added over 5 minutes. After 1.5 hours, the reaction was heated at reflux for 0.5 hours, then cooled to room temperature. The solution was diluted with EtOAc /
Poured into 1N HCl solution. [Note], and the layers were separated. The organic layer was washed with water, saturated Na ₂ CO ₃ solution and brine (4 ×), dried (Na ₂ SO ₄ ) and concentrated under reduced pressure to give the title product (36.3 g, 87% yield). Was.

Step F: Preparation of 4-cyano-3-methoxybenzyl bromide The alcohol from Step E (35.5 g, 218 mm) in 500 mL of dry THF
ol) was cooled to 0 ° C. Triphenylphosphine (85.7 g,
327 mmol), followed by carbon tetrabromide (108.5 g, 327 mmol).
l) was added. The reaction was stirred at 0 ° C. for 30 minutes, then at room temperature for 21 hours. Silica gel (about 300 g) was added and the suspension was concentrated under reduced pressure. The resulting solid was applied to a silica gel chromatography column. Purify by flash chromatography (30% -50% EtOAc / hexane) to give the title product (4.
2 g, 85% yield).

Step G: Preparation of 1- (4-cyano-3-methoxybenzyl) -5- (acetoxymethyl) -imidazole hydrobromide Step F using the procedure outlined in Step C of Example 1. From bromide (21.7 g
, 96 mmol) with the imidazole product from Step B of Example 1 (34.9 g,
(91 mmol) to give the title product. The crude product was triturated with hexane to give the hydrobromide of the title product (19.43 g, 88% yield).

Step H: Preparation of 1- (4-Cyano-3-methoxybenzyl) -5- (hydroxymethyl) -imidazole The title product (19.43 g, 68.1 mmol) was prepared by decomposition. The crude title product was isolated in low yield (11 g, 66% yield). Concentrate the aqueous extract with ¹ H
Solid product (approximately 1%) containing significant amounts of the title product as judged by NMR spectroscopy.
00g).

Step I: Preparation of 1- (4-cyano-3-methoxybenzyl) -5-imidazolecarboxaldehyde Using the procedure outlined in Example 1, Step E, the alcohol from Step H (11 g,
The title product was prepared by oxidizing 45 mmol). The title aldehyde, without further purification, was sufficiently pure white powder to be used in the next step (7.
(4 g, 68% yield).

Step J: Preparation of 1- (3-chlorophenyl) -4- [1- (4-cyano-3-methoxybenzyl) imidazolylmethyl] -2-piperazinone dihydrochloride As outlined in Step H of Example 1. The aldehyde from Step I (85
9 mg, 3.56 mmol) and the amine (hydrochloride) from step K of Example 1 (
The title product was prepared by reductively alkylating 800 mg, 3.24 mmol). Flash column chromatography through silica gel (50%
Purification by 75% acetone _CH 2 Cl _2), the resulting white foam of the title product was added to the dihydrochloride salt as a white powder (743 mg, 45% yield).
FAB ms (m + 1) 437. _{Analysis: C 23 H 23 ClN 5 O} 2 · 2.0HCl · 0.35CH 2 Cl 2 Calculated for: C, 51.97; H, 4.80 ; N, 12.98. Found: C, 52.11; H, 4.80; N, 12.21.

Example 7 1- (3-trifluoromethoxyphenyl) -4- [1- (4-cyano-3-
Methoxybenzyl) imidazolylmethyl] -2-piperazinone dihydrochloride 1- (3-trifluoromethoxyphenyl) -2-piperazinone hydrochloride was prepared from 3-trifluoromethoxyaniline using steps FJ of Example 1. did.
This amine (1.75 g, 5.93 mmol) was combined with the aldehyde from Example I, Step I (1.57 g, 6.52 mmol) using the procedure outlined in Example 1, Step H.
l). Flash column chromatography through silica gel (6
Purification by 0-100% acetone _{CH 2} Cl 2), white powder of the title product by the addition of white foam on its dihydrochloride obtained (1.957g, yield 5
9%). FAB ms (m + 1) 486. _{Analysis: C 24 H 23 F 3 N} 5 O 3 · 2.0HCl · 0.60CH 2 O Calculated for: C, 50.64; H, 4.46 ; N, 12.30. Found: C, 50.69; H, 4.52; N, 12.13.

Example 8 4-[((1- (4-Cyanobenzyl) -5-imidazolyl) methyl) amino] benzophenone Hydrochloride Using the procedure outlined in Step K of Example 1, Aldehyde from Step E (124 mg, 0.588 mmol) and 4-aminobenzophenone (1
The title product was prepared by reductively alkylating 16 mg, 0.588 mmol). Flash column chromatography through silica gel (2% ~
Purification by 6% MeOH / CH ₂ Cl ₂ ) and conversion to the hydrochloride afforded the title product as a white solid (126 mg, 50% yield). FAB ms (m + 1) 393.11. _{Analysis: C 25 H 20 N 5 O} · 1.40HCl · 0.40H 2 O Calculated for: C, 66.62; H, 4.96 ; N, 12.43. Found: C, 66.73; H, 4.94; N, 12.46.

Example 9 N- {1- (4-cyanobenzyl) -1H-imidazol-5-ylmethyl}
-4 (R) -benzyloxy-2 (S)-{N'-acetyl-N'-3-chlorobenzyl} aminomethylpyrrolidine Step A: 4 (R) -hydroxyproline methyl ester 4 (R) in methanol (500 ml) R) -Hydroxyproline (35.12 g)
, 267.8 mmol) was saturated with gaseous hydrochloric acid. The resulting solution was left for 16 hours and the solvent was evaporated under reduced pressure to give the title compound as a white solid. ¹ H NMR CD ₃ OD δ 4.60 (2H, m), 3.86 (3H, s),
3.48 (1H, dd, J = 3.6 and 12.0 Hz), 3.23 (1H, d
, J = 12.0 Hz), 2.43 (1H, m) and 2.21 (1H, m) pp
m.

Step B: Nt-butoxycarbonyl-4 (R) -hydroxyprolinemethyl
Ester CH₂Cl₂4 (R) -Hydroxyproline methyl ester in (500 ml)
(53.5 g, 268 mmol) and trimethylamine (75 ml, 540
mmol) in a solution containing₂Cl₂(75 ml) in di-t-butyl dicar
A solution containing Bonate (58.48, 268 mmol) was added. The resulting mixture
The material was stirred at room temperature for 48 hours. The solution was diluted with 10% aqueous citric acid, saturated NaHCO ₃ Solution coarse, dried (Na₂SO₄) And the solvent was evaporated under reduced pressure. Notation
Was obtained as a yellow oil which was used in the next step without further purification.¹ H NMR CD₃OD δ 4.40 to 4.30 (2H, m), 3.75 (3
H, m), 3.60-3.40 (2H, m), 2.30 (1H, m), 2.05
(1H, m) and 1.55-1.40 (9H, m) ppm.

Step C: Nt-butoxycarbonyl-4 (R) -t-butyldimethylsilyloxyproline methyl ester Nt-butoxycarbonyl-4 (R) -hydroxyproline in CH ₂ Cl ₂ (536 ml) A solution containing methyl ester (65.87 g, 268 mmol) and trimethylamine (41 ml, 294 mmol) was added to CH ₂ Cl ₂ (86
tert-butyldimethylsilyl chloride (42.49 g, 282 mmol
The solution containing 1) was added. The resulting mixture was stirred at room temperature for 16 hours. Wash the solution with 10% aqueous citric acid, saturated aqueous NaHCO ₃ and dry (Na ₂ SO ₄ )
And the solvent was evaporated under reduced pressure. The title compound was obtained as a yellow oil and used in the next step without further purification. ¹ H NMR CD ₃ OD δ 4.60 to 4.40 (2H, m), 3.75 (3
H, m), 3.60 to 3.20 (2H, m), 2.30 to 1.90 (2H, m)
, 1.45 to 1.40 (9H, m), 0.90 to 0.85 (9H, m), 0.1
0-0.00 (6H, m) ppm.

Step D: Nt-butoxycarbonyl-4 (R) -t-butyldimethylsilyloxy-2 (S) -hydroxymethylpyrrolidine Nt-butoxycarbonyl-4 (R) in THF (150 ml) -T-butyldimethyl-silyloxyproline methyl ester (86.65 g, 241 mmol
l) was added to lithium aluminum hydride (THF) under an argon atmosphere.
(247 ml of a 1M solution in 247 mmol) was added over a period of 90 minutes so that the temperature did not exceed 12 ° C. Stirring was continued for 50 minutes, then EtOAc (
(500 ml) was carefully added, followed by sodium sulfate decahydrate (34 g) and the resulting mixture was stirred at room temperature for 16 hours. Anhydrous Na ₂ SO ₄ (34 g)
Was added and the mixture was stirred for a further 30 minutes, then filtered. Solids in EtOAc
(800 ml), the filtrates were combined and the solvent was evaporated under reduced pressure. The title compound was obtained as a colorless oil and used in the next step without further purification.

Step E: Nt-butoxycarbonyl-4 (R) -t-butyldimethylsilyloxy-2 (S) -methanesulfonyloxymethylpyrrolidine Nt-butoxy in CH ₂ Cl ₂ (1 L) Carbonyl-4 (R) -t-butyldimethylsilyloxy-2 (S) -hydroxymethylpyrrolidine (50.0 g,
150.8 mmol) and triethylamine (42.0 ml, 300 mmol)
) Was added to the solution containing methanesulfonyl chloride (12.4 ml, 160 mmol).
) Was added over 5 minutes and stirred for 1 hour. The solvent was evaporated under reduced pressure and EtO
Diluted with Ac (800 ml), successively washed with aqueous citric acid and NaHCO _3. The organic extract was dried (Na ₂ SO ₄ ), evaporated under reduced pressure and the residue was purified by chromatography (SiO ₂ , 15% EtOAc in hexane). The title compound was obtained as a pale yellow solid. FAB mass spectrum, m / z = 410 (M + 1). ¹ H NMR CDCl ₃ δ 4.60-4.00 (4H, m), 3.60-3
. 30 (2H, m), 2.98 (3H, s), 2.05-2.00 (2H, m)
, 1.48 to 1.42 (9H, m), 0.90 to 0.80 (9H, m), 0.1
0-0.00 (6H, m) ppm.

Step F: Preparation of Nt-butoxycarbonyl-4 (R) -t-butyldimethylsilyloxy-2 (S) -azidomethylpyrrolidine In a flask protected with a safety screen, in toluene (250 ml) Nt
-Butoxycarbonyl-4 (S) -t-butyldimethylsilyloxy-2 (S)-
Methane-sulfonyloxymethylpyrrolidine (10.40 g, 25.39 mmol
) And tetrabutylammonium azide (8.18 g, 28.7 mmol) were stirred at 80 ° C. for 5 hours. The reaction was cooled to room temperature and EtOAc (2
50 ml), washed with water and brine, and dried (Na ₂ SO ₄ ). The solvent was evaporated under reduced pressure to give the title compound as a yellow oil, which was used in the next step without further purification. ¹ H NMR CDCl ₃ δ 4.60-3.20 (6H, m), 2.05-1
. 90 (2H, m), 1.47 (9H, s), 0.87 (9H, s) and 0.
10-0.00 (6H, m) ppm.

Step G: Preparation of Nt-butoxycarbonyl-4 (R) -t-butyldimethylsilyloxy-2 (S) -aminomethylpyrrolidine Nt-butoxycarbonyl-4 (in EtOAc (120 ml) R) -t-
Butyldimethylsilyloxy-2 (S) -azidomethylpyrrolidine (9.06 g,
The solution containing 25.39 mmol) was replaced with argon, and 10% palladium on carbon (
1.05 g) was added. The flask was evacuated to 1 atmosphere under a hydrogen atmosphere (49 psi).
Stir for 6 hours. The hydrogen was replaced with argon, the catalyst was filtered off and the solvent was evaporated under reduced pressure. The residue is chromatographed (SiO ₂ , 2.5 in acetonitrile).
５5% saturated NH ₄ OH, gradient elution) to give the title compound as an oil. ¹ H NMR (CDCl ₃ , 400 MHz) δ 4.40 to 2.60 (6H, s)
, 2.05-1.80 (2H, m), 1.46 (9H, s), 1.36 (2H, m).
s), 0.87 (9H, s), 0.10-0.00 (6H, m) ppm.

Step H: Preparation of Nt-Butoxycarbonyl-4 (R) -t-butyldimethylsilyloxy-2 (S)-{N'-3-chlorobenzyl} aminomethylpyrrolidine In methanol (150 ml) 3-chlorobenzaldehyde (1.2 ml, 10
. 6 mmol), a milled 3A molecular sieve (9.5 g) and the amine from step G (3.50 g, 10.6 mmol) at room temperature in sodium cyanoborohydride (11.0 ml of a 1 M solution in THF, 11 .0 mmol) was added. The pH was adjusted to 7 by the addition of glacial acetic acid (0.68 ml, 12 mmol) and the reaction was stirred for 16 hours. The reaction was filtered and the filtrate was evaporated under reduced pressure. The residue was partitioned between EtOAc and saturated NaHCO ₃ solution, the organic extracts were washed with brine, dried (Na ₂ SO ₄ ) and the solvent was evaporated under reduced pressure. The residue was purified by chromatography (SiO ₂ , 2.5% MeOH in CH ₂ Cl ₂ ) to give the title compound as an oil. ¹ H NMR (CDCl ₃ , 400 MHz) δ 7.40 to 7.10 (4H, m)
, 4.36 (1H, s), 4.15 to 3.90 (2H, m), 3.90 to 3.3
0 (2H, m), 2.85 to 2.60 (2H, m), 2.05 to 1.90 (2H
, M), 1.44 (9H, s), 0.87 (9H, s) and 0.06 (6H, s).
m) ppm.

Step I: Nt-butoxycarbonyl-4 (R) -t-butyldimethylsilyl
Xy-2 (S)-{N'-3-chlorobenzyl-N'-acetyl} aminomethyl
Preparation of pyrrolidine CH₂Cl₂(85 ml) and trimethylamine (2.40 ml, 17.0).
mmol) in Nt-butoxycarbonyl-4 (R) -t-butyldimethylamine.
Riloxy-2 (S)-{N'-3-chlorobenzyl} -aminomethylpyrrolidine
(3.80 g, 8.35 mmol) in a solution containing acetyl chloride (0.6
0 ml, 8.44 mmol) was added. Stir the reaction at room temperature for 1 hour and dilute with water.
Mark, CH₂Cl₂Extracted. The extract is washed with brine and dried (Na₂SO₄ ) And the solvent was evaporated under reduced pressure. The residue was chromatographed (SiO₂, CH ₂ Cl₂(10-25% EtOAc in gradient elution).¹ H NMR (CDCl₃, 400 MHz) δ 7.40 to 7.00 (4H, m)
5.10 to 3.00 (8H, m), 2.20 to 1.70 (5H, m), 1.5
0 to 1.30 (9H, m), 0.87 (9H, s) and 0.06 (6H, m)
ppm.

Step J: Nt-butoxycarbonyl-4 (R) -hydroxy-2 (S)-｛
Preparation of N'-3-chlorobenzyl-N'-acetyl {aminomethylpyrrolidine Nt-butoxycarbonyl-4 (R) -t-butyl-dimethylsilyloxy-2 (S)-} in THF (80 ml). To a solution containing N′-3-chlorobenzyl-N′-acetyl {aminomethylpyrrolidine (4.02 g, 8.09 mmol),
At 0 ° C., tetrabutylammonium fluoride (9.00 m of a 1 M solution in THF)
1, 9.00 mmol) was added. The reaction was stirred at 0 ° C. for 1 hour, then at room temperature for 30 minutes. The reaction was quenched by the addition of saturated Na ₄ Cl solution (50 ml) and diluted with EtOAc. Wash the organic extract with brine and dry (Na ₂ S
O ₄ ) and the solvent was evaporated under reduced pressure. The residue was chromatographed (SiO ₂ ,
3 to 5% MeOH in CH ₂ Cl _2, was obtained as a foam of the title compound was purified by gradient elution). ¹ H NMR (CDCl ₃ , 400 MHz) δ 7.40 to 7.00 (4H, m)
5.00 to 4.00 (4H, m), 4.00 to 3.10 (4H, m), 2.3
0-1.60 (5H, m) and 1.50-1.30 (9H, m) ppm.

Step K: Nt-butoxycarbonyl-4 (R) -benzyloxy-2 (S
)-{N'-Acetyl-N'-3-chlorobenzyl} aminomethylpyrrolidine Nt-butoxycarbonyl-4 (S) -hydroxy- in DMF (9 ml).
2 (S)-{N'-acetyl-N'-3-chlorobenzyl} aminomethylpyrroli
Gin (701 mg, 1.83 mmol) in a solution containing sodium hydride at 0 ° C.
(110 mg of a 60% dispersion in mineral oil, 2.75 mmol) was added. 15 minutes later,
Benzyl bromide (0.435 ml, 3.66 mmol) was added and the reaction was allowed to stand at room temperature.
Stirred for 16 hours. The reaction solution is saturated NaHCO₃Quench with solution (2ml) and vinegar
Extracted with ethyl acid. Wash the organic extract with brine, dry (Na₂SO₄)
The solvent was evaporated under reduced pressure. The residue was chromatographed (SiO₂, CH₂Cl ₂ 25-50% EtOAc in EtOAc, gradient elution) to give the title compound
Was obtained as a foam.

Step L: 4 (S) -benzyloxy-2 (S)-{N′-acetyl-N′-3-
Chlorobenzyl @aminomethylpyrrolidine hydrochloride The product from step K (0.834 g, 1.76 m) in EtOAc (25 ml)
mol.) was saturated at 0 ° C. with gaseous hydrogen chloride. The resulting solution was left at room temperature for 30 minutes. The solvent was evaporated under reduced pressure to give the title compound as a white solid.

Step M: Preparation of 1H-imidazole-4-acetic acid methyl ester hydrochloride 1H-imidazole-4-acetic acid hydrochloride (4.0 ml) in methanol (100 ml).
(0 g, 24.6 mmol) was saturated with gaseous hydrogen chloride. The resulting solution was left at room temperature (RT) for 18 hours. The solvent was evaporated under reduced pressure to give the title compound as a white solid. ¹ H NMR (CDCl ₃ , 400 MHz) δ 8.85 (1 H, s), 7.45
(1H, s), 3.89 (2H, s) and 3.75 (3H, s) ppm.

Step N: Preparation of 1- (triphenylmethyl) -1H-imidazol-4-ylacetic acid methyl ester The product from Step M (2) in dimethylformamide (DMF) (115 ml)
To a solution containing 4.85 g (0.141 mol), triethylamine (57.2 m
1, 0.412 mol) and triphenylmethyl bromide (55.3 g, 0.17
1 mol) was added and the suspension was stirred for 24 hours. After this time, the reaction mixture was diluted with ethyl acetate (EtOAc) (1 L) and water (350 ml). The organic phase was washed with saturated aqueous NaHCO ₃ (350 ml), dried (Na ₂ SO ₄ ) and evaporated under reduced pressure. Flash chromatography the residue was obtained (SiO _2, 0 to 100% ethyl acetate in hexanes, gradient elution) The title compound was purified by as a white solid. ¹ H NMR (CDCl ₃ , 400 MHz) δ 7.35 (1 H, s), 7.31
(9H, m), 7.22 (6H, m), 6.76 (1H, s), 3.68 (3H
, S) and 3.60 (2H, s) ppm.

Step O: [1- (4-Cyanobenzyl) -1H-imidazol-5-yl] vinegar
Preparation of acid methyl ester The product from Step N (8.00 g, 20.9 g) in acetonitrile (70 ml)
bromo-p-tolunitrile (4.10 g, 20.92).
mmol) and heated at 55 ° C. for 3 hours. After this time, bring the reaction to room temperature.
Upon cooling, the resulting imidazolium salt (white precipitate) was collected by filtration. The filtrate
Heated at 55 ° C. for 18 hours. The reaction mixture was cooled to room temperature and evaporated under reduced pressure
. EtOAc (70 ml) was added to the residue and the resulting white precipitate was collected by filtration.
Was. The precipitated imidazolium salt was combined and suspended in methanol (100 ml).
And refluxed for 30 minutes. After this time, the solvent was removed under reduced pressure and the resulting residue was
Suspended in EtOAc (75 ml) and the solid was isolated by filtration and washed (E
tOAc). NaHCO₃Aqueous solution (300 ml) and CH₂Cl ₂ (300 ml) and stirred at room temperature for 2 hours. The organic layer was separated and dried (N
a₂SO₄) And evaporated under reduced pressure to give the title compound as a white solid.¹ H NMR (CDCl₃, 400 MHz) δ 7.65 (1H, d, J = 8 Hz)
), 7.53 (1H, s), 7.15 (1H, d, J = 8 Hz), 7.04 (1
H, s), 5.24 (2H, s), 3.62 (3H, s) and 3.45 (2H
, S) ppm.

Step p: Preparation of (1- (4-cyanobenzyl) -1H-imidazol-5-yl) ethanol The ester from Step O (1.50 g, 5.88 m) in methanol (20 ml)
mol) at 0 ° C. with sodium borohydride (1.0 g, 26.3 mm).
ol) was added in portions over 5 minutes. The reaction was stirred at 0 ° C. for 1 hour, then at room temperature for another 1 hour. The reaction was quenched by the addition of saturated Na ₄ Cl solution and the methanol was evaporated under reduced pressure. The residue was partitioned between EtOAc and saturated NaHCO ₃ solution, and the organic extract was dried (Na ₂ SO ₄ ) and evaporated under reduced pressure. The residue chromatographed to give (SiO _2, 4 to 10% methanol in methylene chloride, gradient elution) The title compound was purified by a solid. ¹ H NMR CDCl ₃ δ 7.64 (2H, d, J = 8.2 Hz), 7.5
7 (1H, s), 7.11 (2H, d, J = 8.2 Hz), 6.97 (1H, s)
), 5.23 (2H, s), 3.79 (2H, t, J = 6.2 Hz) and 2.
66 (2H, t, J = 6.2 Hz) ppm.

Step Q: 1- (4-Cyanobenzyl) -imidazol-5-yl-ethylmethanesulfonate (1- (4-cyanobenzyl) -1H-imidazol-5-yl in methylene chloride (6.0 ml) ) -A solution containing ethanol (0.500 g, 2.20 mmol) was treated at 0 ° C. with Hunig's base (0.460 ml, 2.64 mmol) and methanesulfonyl chloride (0.204 ml, 2.64 mmol). After 2 hours, the reaction was quenched by the addition of saturated NaHCO ₃ solution (50 ml), the mixture was extracted with methylene chloride (50 ml), dried (Na ₂ SO ₄ ) and the solvent was evaporated under reduced pressure. The title compound was used without further purification. ¹ H NMR CDCl ₃ δ 7.69 (1H, s), 7.66 (2H, d, J
= 8.2 Hz), 7.15 (2H, d, J = 8.2 Hz), 7.04 (1H, s)
), 5.24 (2H, s), 4.31 (2H, t, J = 6.7 Hz), 2.96
(3H, s) and 2.88 (2H, t, J = 6.6 Hz) ppm.

Step R: N {1- (4-cyanobenzyl) -1H-imidazol-5-ylethyl} -4 (R) -benzyloxyoxy-2 (S)-{N′-acetyl-N′-
3- (Chlorobenzyl) -aminomethylpyrrolidine 4 (R) -benzyloxy-2 (S)-{N'-acetyl-N'-3-chlorobenzyl-aminomethyl} pyrrolidine (199 mg, 0
. 486 mmol), the methylate from step Q (140 mg, 0.458 mmol)
l), a mixture containing potassium carbonate (165 mg, 1.19 mmol) and sodium iodide (289 mg, 1.93 mmol) was heated at 55 ° C. for 16 hours.
The cooled mixture was diluted with EtOAc, washed with NaHCO ₃ solution and brine, dried (Na ₂ SO ₄ ) and the solvent was evaporated under reduced pressure. Preparative HPLC of the residue
C-18, water / acetonitrile with 0.1% TFA 95: 5 to 5:95, gradient elution). The title compound was obtained as a white solid after lyophilization. _{Analysis: C 34 H 36 N 5 O} 2 Cl 3.00TFA, calculated for _{0.85H 2 O: C, 51.14;} H, 4.37; N, 7.45. Found: C, 51.15; H, 4.42; N, 6.86. _{FAB HRMS C 34 H 37 N 5} O 2 exact mass calculated for Cl 582.263579 ^(NH +). Found: 582.2263900.

Example 10 In Vitro Inhibition Transferase Assay. Prenyl protein transferase activity
Saying is performed at 30 ° C. unless otherwise specified. A typical reaction is (final volume 50 μL
As): [³H] farnesyl diphosphate or [³H] geranylgeranyl
Acid, Ras protein, 50 mM HEPES, pH 7.5, 5 mM MgCl ₂ 5 mM dithiothreitol, 10 μM ZnCl₂, 0.1% polyethylene
Glycol (PEG) (molecular weight 15,000 to 20,000) and isoprene
Nil protein transferase. 10 mM glycerol phosphate or 5
Prepared anions such as mM ATP may also be added to the assay medium. In the assay
The FPTase used is described by Omer, C .; A. Kral, A .; M. , Diehl
, R .; E. FIG. Prendergast, G .; C. Powers, S .; , All
en, C.I. M. Gibbs, J .; B. And Kohl, N .; E. FIG. Written, (199
3 years) Biochemistry 32: 5167-5176.
It is prepared by such recombinant expression. Geranylgeranyltan used in the assay
Pak transferase type I is described in US Pat.
It is prepared as described in US Pat. Assay mixing in the absence of enzyme
Thermal pre-equilibration of the product followed by the addition of isoprenyl protein transferase
To initiate the reaction and intermittent by the addition of 1 M HCl in ethanol (1 mL)
(Typically every 15 minutes). Leave the quenched reaction for 15 minutes
(For completion of the precipitation process). After adding 2 mL of 100% ethanol, the reaction solution was
Filter under reduced pressure through a Whatman GF / C filter. 10 filters
Wash 4 times with 2 mL of 0% ethanol, mix with scintillation fluid (10 mL)
And then at the Beckman LS3801 scintillation counter.
Und

For inhibition studies, the inhibitors are prepared as concentrated solutions in 100% dimethyl sulfoxide and then assayed as described above, except that they are diluted 20-fold into the enzyme assay mixture. The substrate concentrations for the determination of the IC ₅₀ of the inhibitor are given below: FTa
se, 650 nM Ras-CVLS (SEQ ID NO: 2), 100 nM farnesyl diphosphate, GGPase-I, 500 nM Ras-CAIL ((SEQ ID NO: 3), 100 nM geranylgeranyl diphosphate.

Example 11 Modified In Vitro GGTase Inhibition Assay A modified geranylgeranyl protein transferase inhibition assay is performed at room temperature. A typical reaction is (assuming a final volume of 50 μL): [ ³ H] geranylgeranyl diphosphate, biotinylated Ras peptide, 50 mM HEPES, pH 7.5,
Regulating anions (eg, 10 mM glycerophosphate or 5 mM ATP), 7
mM MgCl ₂ , 10 μM ZnCl ₂ , 0.1% PEG (molecular weight 15,000
0-20,000), 2 mM dithiothreitol, and geranylgeranyl protein transferase type I (GGTase-I). GGTase-I type enzyme used in the assay is described in U.S. Pat. 5,4
70, 832. The Ras peptide is derived from the K4B-Ras protein and has the following sequence: biotinyl-GKKKKKKSKTKTK
CVIM (single amino acid code) (SEQ ID NO: 13). The reaction was started by the addition of GGTase, pH 4, 0, containing 50 mM EDTA and 0.5% BSA.
. Streptavidin SPA beads (Scintil) in 2M sodium phosphate
relation Proximity Assay beads, Amersha
m) (3 μg / mL) containing 200 μL of a 3 mg / mL suspension.
Every minute). The quenched reaction is left for 2 hours before packing
d Analyzed on TopCount scintillation counter.

For inhibition studies, the inhibitors are prepared as concentrated solutions in 100% dimethyl sulfoxide and then assayed as described above, except that they are diluted 25-fold into the enzyme assay mixture. For inhibition studies with delayed binding inhibitors, GGTase and inhibitor were preincubated for 1 hour and F. Morrison, C.I. T. W
alsh, Adv. Enzymol & Related Areas Mo
l. Biol. 61, 201-301 (1988), the reaction is started by adding a peptide substrate. The IC ₅₀ is determined using the Ras peptide near the _{K M} concentration. Enzyme and substrate concentrations to determine the IC ₅₀ of the inhibitor are: 75 pM GGTase-I, 1.6 μM Ras peptide, 100 nM geranylgeranyl diphosphate.

[0304] In addition, enzymatic _{K i} values for the inhibition of GGPTase-I is, I. H. Seg
el ("Enzyme Kinetics", pp. 342-345; Wiley
And Sons, New York, N.W. Y. (1975) and references cited therein).

Example 13 Cell Lines In Vitro ras Prenylation Assay The cell lines used in this assay were Rat1 or NIH3T3 cells transformed with the virus H-ras; the C-terminal hypervariable region of vH-ras. N
An N-ras chimeric gene replaced with the corresponding region from the -ras gene; or R
as-CVLL (SEQ ID NO: 1), a virus-H-ras mutation in which the C-terminal exon encodes leucine instead of serine, thereby rendering the encoded protein a substrate for geranylgeranylation by GGTase-I Consists of seeds. Assays can also be performed using cell lines transformed with human H-ras, N-ras or K4B-ras. The assay is essentially a DeClue, J
. E. FIG. Et al., Cancer Research 51: 712-717 (1991). Cells in a 10 cm Petri dish with 50-75% confluence are treated with the test compound (final concentration of solvent, methanol or dimethylsulfoxide 0.1%). After 4 hours at 37 ° C., cells were washed with 10% normal DME
M, 2% fetal calf serum, 400 μCi [ ³⁵ S] methionine (1000 Ci / m
mol) and test compound in 3 ml of methionine-free DMEM. Treatment with lovastatin, a compound that inhibits Ras processing in cells by inhibiting the rate limiting step in the isoprenoid biosynthetic pathway (
Hancock, J. et al. F. Authors, Cell, 57: 1167 (1989); DeClue, J. et al. E. FIG. Authors, Cancer Res, 51: 712 (1991); Sinensky, M .; J. et al. Biol. Chem, 2nd
65: 19937 (1990)) serves as a positive control in this assay. After an additional 20 hours, the cells were washed with 1 ml of lysis buffer (1% NP40 / 20 mM HEPES, pH 7.5 / 5 mM MgCl ₂ /
1 mM DTT / 10 mg / ml aprotinin / 2 mg / ml leupeptin / 2 mg / ml antipain / 0.5 mM PMSF) and dissolve the lysate in 1
Remove by centrifugation at 00,000 × g for 45 minutes. Also, 4 hours after the addition of the labeling medium, the medium is removed, the cells are washed and 3 ml of medium containing the same or a different test compound is added. After an additional 16 hours of incubation, lysis is performed as described above. Lysates containing the same number of acid precipitation counts were added to IP buffer (DT
Lysis buffer lacking T) to make 1 ml, ras-specific monoclonal antibody Y13-
259 (Furth, ME et al., J. Virol. 43: 294-30).
4 (1982)). After incubating the antibody at 4 ° C. for 2 hours, 200 μl of a 25% suspension of protein A-sepharose coated with rabbit anti-rat IgG is added for 45 minutes. The immunoprecipitate was subjected to SDS-PA
IP buffer (20 nM HEPES, pH 7.5 / 1 mM EDTA / 1% Trito) boiled in GE sample buffer and loaded with 13% acrylamide gel
n X-100.0.5% deoxycholate / 0.1% / SDS / 0.1M N
aCl) 4 times. When the tip of the dye reaches the bottom, the gel is fixed and Enli
Immerse in ghtening, dry and take radiographs. The intensity of the bands corresponding to the prenylated and non-prenylated Ras proteins is compared to determine the percent inhibition of prenyl transfer to the protein.

Example 14 Construction of SEAP Reporter Plasmid pDSE100 The SEAP reporter plasmid pDSE100 was assembled by ligating a restriction fragment containing the SEAP coding sequence to plasmid pCMV-RE-AKI. The SEAP gene is expressed in plasmid pSEAP2-Basic (C
lontech, Palo Alto, CA). Plasmid pCMV-RE-AKI was assembled by Deborah Jones (Merck) and derived from the cytomegalovirus early promoter "CAT".
-Contains 5 consecutive copies of the "stain symmetry reaction element" cloned upstream of the "TATA" sequence.

The plasmid pDSE100 was assembled as follows. A restriction fragment encoding the SEAP coding sequence was excised from plasmid pSEAP2-Basic using restriction enzymes EcoR1 and HpaI. The end of the linear DNA fragment was E. coli DNA polymerase I was filled with the Klenow fragment. "Blunt end" DNA containing the SEAP gene was isolated by electrophoresis of the digest in an agarose gel and cutting out the 1694 base pair fragment. The vector plasmid pCMV-RE-AKI was linearized with the restriction enzyme Bgl-II and the ends were filled with Klenow DNA polymerase I. SEAP
DNA is blunt end ligated into the pCMV-RE-AKI vector and the ligation product is DH5-αE. E. coli cells (Gibco-BRL). Transformants were screened for the appropriate insert and a genetic map was created for restriction fragment orientation. Properly oriented recombination constructs were sequenced beyond the cloning junction to confirm the correct sequence. The resulting plasmid is downstream of the DSE and CAT-TATA promoter elements and BGHpo.
Contains the SEAP coding sequence upstream of the ly-A sequence.

Alternative Assembly of SEAP Reporter Plasmid pDSE101 The SEAP reporter plasmid, pDSE101, is also assembled by ligating a restriction enzyme containing the SEAP coding sequence to plasmid pCMV-RE-AKI. The SEAP gene is derived from the plasmid pGEM7zf (-) / SEAP.

Plasmid pDSE101 was assembled as follows: a restriction fragment containing a portion of the SEAP gene coding sequence was excised from plasmid pGEM7zf (-) / SEAP using restriction enzymes ApaI and KpnI. The ends of the linear DNA fragment were It was pushed back with the Klenow fragment of E. coli DNA polymerase I. "Blunt end" DNA containing the truncated SEAP gene was isolated by electrophoresis of the digest in an agarose gel and excising the 1910 base pair fragment. This 1910 base pair fragment was ligated into the plasmid pCMV-RE-AKI cut with Bgl-II and E. coli Klenow fragment DNA polymerase. Recombinant plasmids were screened for insert orientation and sequenced through the junction. Plasmid pCMV-R
E-AKI was prepared using plasmid pCMVIE-AKI-DHFR (Whang, Y.,
Silverklang, M .; Morgan, A .; Munshi, S .; , L
any, A .; B. Ellis, R .; W. And Kieff, E .; (19
1987). Virol. 61, pp. 1796-1807), from DHF
Induced by removing the EcoRI fragment containing the R and neomycin markers. Five copies of the fos promoter formation reaction element were described previously (J.
ones, R.E. E. FIG. Defeo-Jones, D .; McAvoy, E .; M.
Vuolo, G .; A. Wegrzyn, R .; J. , Haskell, K
. M. And Oliff, A .; Written (1991) Oncogene, Volume 6, 7
(Pages 45 to 751) to form plasmid pCMV-RE-AKI.

The plasmid pGEM7zf (−) / SEAP was assembled as follows.
The SEAP gene was transformed into a human placenta cDNA library (C
PCR (Lontech).

[0311]

Embedded image Using N-terminal oligos (SEQ ID NO: 4 and SEQ ID NO: 5), Ec
A 1560 bp N-terminal PCR product was generated containing the oRI and HpaI restriction sites. The antisense N-terminal oligo (SEQ ID NO: 5) introduces an internal translation stop codon in the SEAP gene along the HpaI site. C-terminal oligo (
SEQ ID NO: 6 and SEQ ID NO: 7) were used to amplify a 412 bp C-terminal PCR product containing HpaI and HindIII restriction sites. The sense strand C-terminal oligo (SEQ ID NO: 6) introduces an internal stop codon as well as an HpaI site. Next, the N-terminal amplicon was digested with EcoRI and HpaI and the C-terminal amplicon was digested with HpaI and HindIII. SEAP
Two fragments containing each end of the gene were isolated by electrophoresis of the digest in an agarose gel and isolating the 1560 and 412 base pair fragments. These two fragments were then restriction digested with EcoRI and HindIII and the vector pGEM7zf (-) (-) () isolated in an agarose gel.
Promega). The resulting clone pGEM7zf (-) /
SEAP contains the coding sequence for the SEAP gene from amino acids.

Assembly of the constitutively expressed SEAP plasmid pCMV-SEAP-A. By placing an encoding sequence. The expression plasmid contains the 3 'untranslated region of the bovine growth hormone gene 3' to the CMV intron A region 5 '
Also includes the SEAP gene.

The plasmid pCMVIE-AKI-DHFR containing the CMV early promoter (
Whang, Y .; Silverberg, M .; Morgan, A .; , Mu
nshi, S .; Lenny, A .; B. Ellis, R .; W. , And Kie
ff, E. Written (1987) Virol. 61: 1796-180
7) was cut with EcoRI resulting in two fragments. Vector fragments were isolated by agarose gel electrophoresis and religated. The resulting plasmid is called pCMV-AKI. Next, the cytomegalovirus intron A nucleotide sequence was inserted downstream of the CMV-IEI promoter in pCMV-AKI. The intron A sequence was isolated from a genomic clone bank and pBR322
Subcloned into plasmid p16T-286. Intron A
The sequence was mutated at nucleotide 1856 (Chapman, BS, Th.
ayer, R .; M. , Vincent, K .; A. And Haigwood, N .;
L. Author, Nuc. Acids Res. 19, pp. 3979-3986), the SacI restriction site was removed using site-directed mutagenesis. The mutated intron A sequence was subjected to PCR from plasmid p16T-287 using the following oligos.

[0314]

Embedded image These two oligos generate a 991 base pair fragment with a SacI site incorporated by the sense oligo and a Bgl-II fragment incorporated by the antisense oligo. The PCR fragment is trimmed with SacI and Bgl-II and isolated on an agarose gel. Vector pCMV-AK
I is cut with SacI and Bgl-II and the larger vector fragment is isolated by agarose gel electrophoresis. The two gel isolated fragments were ligated at the SacI and Bgl-II sites, respectively, to form plasmid pCMV-AKI-I.
Form nA.

The DNA sequence encoding the truncated SEAP gene is inserted into the pCMV-AKI-InA plasmid at the Bgl-II site of the vector. The SEAP gene was transformed with the plasmid pGEM7zf (-) / S using EcoRI and HindIII.
Cut out from EAP (described above). This fragment is filled with Klenow DNA polymerase and a 1970 base pair fragment isolated from the vector fragment by agarose gel electrophoresis. The pCMV-AKI-InA vector is prepared by digesting with Bgl-II and filling the ends with Klenow DNA polymerase. The final construct was constructed using SECM in the pCMV-AKI-InA vector.
Generated by blunt end ligation of the AP fragment. Transformants were screened for the appropriate insert and then mapped for restriction fragment orientation. Properly oriented recombination constructs were sequenced beyond the cloning junction to confirm the correct sequence. The resulting plasmid was pCMV-S
It is called EAP-A (deposited with the ATCC under the Budapest Treaty on August 27, 1998 and designated as ATCC) and is a cytomegalovirus early promoter IE.
It contains a modified SEAP sequence downstream of the -1 and intron A sequences and upstream of the bovine growth hormone poly-A sequence. The plasmid constitutively expresses SEAP when transfected into mammalian cells.

Another construct constitutively expressing the SEAP plasmid pCMV-SEAP-B An expression plasmid that constitutively expresses the SEAP protein is located downstream of the cytomegalovirus (CMV) IE-1 promoter and the bovine growth hormone gene. 3 'of
It can be formed by placing a sequence encoding a truncated SEAP gene upstream of the untranslated region.

The plasmid pCMVIE-AKI-DHFR containing the CMV early promoter and the bovine growth hormone poly-A sequence (Whang, Y., Silverkl
ang, M .; Morgan, A .; Munshi, S .; Lenny, A .; B
. Ellis, R .; W. And Kieff, E .; Written (1987) Vi
rol. 61: 1796-1807) can be cut with EcoRI to generate two fragments. Vector fragments can be isolated and religated by agarose electrophoresis. The resulting plasmid was pCMV-
Called AKI. The DNA sequence encoding the truncated SEAP gene can be inserted into pCMV-AKI plasmid at the unique Bgl-II in the vector. The SEAP gene was transformed into plasmid p using EcoRI and HindIII.
GEMzf (-) / SEAP (described above). This fragment is
Fill with lnow DNA polymerase and a 1970 base pair fragment isolated from the vector fragment by agarose gel electrophoresis. pCMV-
The AKI vector is prepared by digesting with Bgl-II and filling the ends with Klenow DNA polymerase. The final construct is blunt-end ligated to the SEAP fragment in the vector, It is caused by altering the binding reaction into E. coli DH5α cells. Transformants were screened for the appropriate insert and then mapped for restriction fragment orientation. Properly oriented recombination constructs were sequenced beyond the cloning junction to confirm the correct sequence. The resulting plasmid, called pCMV-SEAP-B, is downstream of the cytomegalovirus early promoter IE1 and bovine growth hormone poly-A.
Contains a modified SEAP sequence upstream of the sequence. The plasmid constitutively expresses SEAP when transfected into mammalian cells.

Cloning of myristylated virus H-ras expression plasmid pSMS600 The DNA fragment containing the virus-H-ras was ligated with plasmid HB-11 (August 27, 1997 by the Budapest Treaty) using the following oligos: Originally ATC
C, and designated ATCC 209, 218).

[0319]

Embedded image

A sequence encoding the first 15 amino acids of the v-src gene containing the myristylation site is incorporated into the sense strand oligo. The sense strand oligo starts at 5 '
Optimize the “Kozak” translation start sequence to the G start site. Virus-rasC
-To prevent prenylation at the terminus, cysteine 186 is mutated to serine by replacing the C residue with a G residue in the C-terminal antisense oligo. The PCR primer oligo introduces an XhoI site at the 5 'end and an XbaI site at the 3' end. XhoI and XbaI fragments were converted to XhoI
And XbaI digested mammalian expression plasmid pCI (Promega)
Can be combined inside. Thereby, the recombined myr-virus-H-ra
A plasmid pSMS600 is obtained in which the s gene is constitutively transcribed from the CMV promoter of the pCI vector.

Cloning of the virus-H-ras-CVLL expression plasmid pSMS601 Virus-H-ras with a C-terminal sequence encoding amino acid CVLL
Clones can be cloned from plasmid "HB-11" by PCR using the following oligos.

[0322]

Embedded image

The sense strand oligo optimizes the “Kozak” sequence and adds XhoI. The antisense strand mutates serine 189 to leucine and adds XbaI. PCR
The fragment is prepared with XhoI and XbaI and inserted into the XhoI-XbaI cut vector pCI (Promega). Thereby, the mutant virus -H-
A plasmid pSMS601 is obtained in which the rasCVLL gene is constitutively transcribed from the CMV promoter of the pCI vector.

Cloning of Cell-H-ras-Leu61 Expression Plasmid pSMS620 The human cell-H-ras gene can be PCR from a human cerebral cortex cDNA library (Clontech) using the following oligonucleotide primers.

[0325]

Embedded image

The primers amplify the c-H-Ras encoding DNA fragment, optimize the “Kozak” translation initiation sequence, an EcoRI site at the N-terminus and a C
-Contributes to a Sal I site at the end. After adjusting the ends of the PCR product with EcoRI and SalI, the cH-Ras fragment was ligated with EcoRI-Sa
It can be ligated into the lI-cut mutagenesis vector pAlter-1 (Promega). Mutation of glutamine-61 to leucine can be achieved using the manufacturer's protocol and the following oligonucleotides.

[0327]

Embedded image

After selection and sequencing for exact nucleotide substitutions, the mutated c-H
-Ras-Leu61 was converted to pAlter using EcoRI and SalI.
-1 vector can be excised and ligated directly into the vector pCI (Promega) digested with EcoRI and SalI. The new recombination plasmid pSMS620 transcribes c-H-ras-Leu61 constitutively from the CMV promoter of the pCI vector.

Cloning of cN-ras-Val-12 Expression Plasmid pSMS630 The human c-N-ras gene can be PCR from a human cerebral cortex cDNA library (Clontech) using the following oligonucleotide primers: .

[0330]

Embedded image

The primers amplify the c-H-Ras encoding DNA fragment, optimize the “Kozak” translation initiation sequence, an EcoRI site at the N-terminus and a C
-Contributes to a Sal I site at the end. After adjusting the ends of the PCR product with EcoRI and SalI, the cN-Ras fragment was ligated with EcoRI-Sa
It can be ligated into the lI-cut mutagenesis vector pAlter-1 (Promega). The glycine-12 to valine mutation can be achieved using the manufacturer's protocol and the following oligonucleotides.

[0332]

Embedded image

After selection and sequencing for exact nucleotide substitutions, the mutated c-N
-Ras-Val-12 was converted to pAlte using EcoRI and SalI.
It can be excised from the r-1 vector and ligated directly into the vector pCI (Promega) digested with EcoRI and SalI. The new recombination plasmid pSMS630 transcribes cN-ras-Val-12 constitutively from the CMV promoter of the pCI vector.

Cloning of c-K4B-ras-Val-12 Expression Plasmid pSMS640 The human c-K4B-ras gene can be PCR-PCR from a human cerebral cortex cDNA library (Clontech) using the following oligonucleotide primers: .

[0335]

Embedded image

The primers amplify the c-K4B-Ras encoding DNA fragment and contribute an optimized “Kozak” translation initiation sequence, a Kpn I site at the N-terminus and a Sal I site at the C-terminus. Insert the end of the PCR product with Kpn I
After preparation with SalI and SalI, the c-K4B-Ras fragment was converted to Kpn
It can be ligated into the I-Sal I cleavage mutagenesis vector pAlter-1 (Promega). The cysteine-12 to valine mutation can be achieved using the manufacturer's protocol and the following oligonucleotides.

[0337]

Embedded image

After selection and sequencing for exact nucleotide substitutions, the mutated cK
4B-ras-Val-12 was converted to pAl using Kpn I and Sal I.
It can be excised from the ter-1 vector and ligated directly into the vector pCI (Promega) digested with Kpn I and Sal I.
The new recombination plasmid transcribes c-K4B-ras-Val-12 constitutively from the CMV promoter of the pCI vector.

Cloning of cK-ras4A-Val-12 Expression Plasmid pSMS650 The human c-K4A-ras gene can be PCR-PCR from a human cerebral cortex cDNA library (Clontech) using the following oligonucleotide primers: .

[0340]

Embedded image

The primers amplify the c-K4A-Ras encoding DNA fragment and contribute an optimized “Kozak” translation initiation sequence, a Kpn I site at the N-terminus and a Sal I site at the C-terminus. Insert the end of the PCR product with Kpn I
After preparation with SalI and SalI, the CK-ras4A fragment was converted to Kpn
It can be ligated into the I-Sal I cleavage mutagenesis vector pAlter-1 (Promega). The cysteine-12 to valine mutation can be achieved using the manufacturer's protocol and the following oligonucleotides.

[0342]

Embedded image

After selection and sequencing for exact nucleotide substitutions, the mutated cK
4A-ras-Val-12 was converted to pAl using Kpn I and Sal I.
It can be excised from ter-1 and ligated directly into the vector pCI (Promega) digested with Kpn I and Sal I. The new recombination plasmid pSMS650 constitutively transcribes c-K4A-ras-Val-12 from the CMV promoter of the pCI vector.

SEAP Assay Human C33A cells (human epithelial carcinoma-ATTC harvest) were isolated from DMEM + 10% fetal calf serum + 1 × Pen / Strep + 1 × glutamine + 1 × NEAA.
Inoculate into 0 cm tissue media plate. Cells are grown at 37 ° C. in a 5% CO ₂ atmosphere until 50-80% confluency is reached.

Transient transfections are performed by the CaPO ₄ method (Sambrook et al., 1989). That is, the expression plasmid for H-ras, N-ras, K-ras, Myr-ras or H-ras-CVLL is co-precipitated with the DSE-SEAP reporter construct. (If cells were transfected with the pCMV-SEAP plasmid, the ras expression plasmid is not included.) For 10 cm plates, Ca
Add 600 μl of the Cl ₂ -DNA solution to 600 μl of 2 × HBS buffer with stirring to obtain 1.2 ml of settling solution (see method below). This is at room temperature for 20-30
Leave for a minute. During sedimentation, the medium of C33A cells is changed to DMEM (phenol-free, Gibco cat. No. 31053-028) + 0.5% charcoal fragment calf serum + 1 × (Pen / Strep, glutamine and non-essential amino acids). It was added dropwise and CaPO _4-DNA sediment cells are dispersed gently vibrate the plate. DNA uptake is allowed to proceed for 5-6 hours at 37 ° C. in a 5% CO ₂ atmosphere.

Following the DNA incubation period, cells are washed with PBS and trypsinized with 1 ml of 0.05% trypsin. 1 ml of cells treated with trypsin
l of phenol red-free DMEM + 0.2% charcoal fragment calf serum + 1 × (Pe
n / Strep, glutamine and non-essential amino acids). The transfected cells were placed in a 96-well microplate (100 μl / well) to which 100 μl of the drug diluted in the medium had been previously added. 200μ final volume per well
1 and each drug concentration is repeated three times in the range of half logarithmic steps.

Incubation of cells and drug is for 36 hours at 37 ° C. under a CO ₂ atmosphere. At the end of the incubation period, the cells are examined microscopically for evidence of cell fatigue. Next, a medium containing secreted alkaline phosphatase (100 μm) was used.
1 is removed from each well, transferred to a microtubule array and heat treated at 65 ° C. for 1 hour to inactivate endogenous alkaline phosphatase (but not thermostable secretory phosphatase).

The fluorescent reagent CSPD® (Tropix, Bedford, Mass.
) Is used to assay the heat-treated medium for alkaline phosphatase by a fluorescence assay. 50 μl of medium are combined with 200 μl of CSPD and incubated at room temperature for 60 minutes. Luminescence is monitored using a ML2200 microplate fluorometer (Dynatech). Luminescence reflects the level of activation of the fos reporter construct by transiently expressed proteins.

DNA-CaPO ₄ precipitation for 10 cm plate of cells Ras expression plasmid (1 μg / μl) 10 μl DSE-SEAP plasmid (1 μg / μl) 2 μl sheared calf thymus DNA (1 μg / μl) 8 μl 2MCaCl ₂ 74 μl dH ₂ O 506 μl 2 × HBS Buffer 280 mM NaCl 10 mM KCl 1.5 mM Na ₂ HPO ₄ 2H ₂ O 12 mM dextrose 50 mM HEPES Final pH = 7.05

Luminescence buffer (26 ml) Assay buffer 20 ml Emerald Reagent ^™ (manufactured by Tropix) 2.5 ml 100 mM homoarginine 2.5 ml CSPD Reagent® (manufactured by Tropix) 1.0 ml

[0351] Assay Buffer 0.05 M Na ₂ CO ₃ to a pH was added to 0.05M _Na 2 HCO ₃ 9.
Make 5

Make up to 1 mM in MgCl ₂ .

Example 14 Cloning of another expression plasmid myristylated c-H-ras expression plasmid pSMS621 PCR of a myristylated c-H-ras-Leu-61 expression plasmid from plasmid pSMS620 (supra) using the following primers: Can be cloned.

[0354]

Embedded image The sequence encoding the first 15 amino acids of the v-src gene including the myristylation site is incorporated into the sense strand oligo. The sense strand oligo starts at 5 '
The “Kozak” translation start sequence up to the G start site is also optimized. v-Ras C-
Cysteine 186 is mutated to serine by replacing the C residue with a G residue in the C-terminal antisense oligo to prevent prenylation at the terminus. The PCR primer oligo has an EcoRI site at the 5 'end and a Sal
An I site is introduced at the 3 'end. The EcoRI-Sal I fragment was
The mammalian expression plasmid pCI (Prom) digested with oRI and SalI
ega). Thereby, the recombined myr-cH-
A plasmid pSMS621 is obtained in which the ras-Ser-186 gene is constitutively transcribed from the CMV promoter of the pCI vector.

Cloning of cH-ras-CVLL Expression Plasmid pSMS622 The cH-ras clone with the C-terminal sequence encoding amino acid CVLL was cloned into plasmid pSMS620 (described above) by PCR using the following oligos: Can be cloned from

Embedded image

The sense strand oligo optimizes the “Kozak” sequence and adds an EcoRI site. The antisense strand mutates serine 189 to leucine and adds a Sal I site. The PCR fragment was prepared with EcoRI and SalI and EcoR
It can be ligated into the I-Sal I digestion vector pCI (Promega). As a result, the mutated cH-ras-Leu61-CVLL gene was transformed into pC
Plasmid pSM constitutively transcribed from the CMV promoter of the I vector
S622 is obtained.

Example 15 In Vivo Tumor Growth Inhibition Assay (Nude Mice) The in vivo effect as an inhibitor of cancer cell growth can be determined by several protocols well known in the art. Examples of such in vivo effects studies are described in N.W. E. FIG. Kohl et al. (Nature Medicine, 1:79)
2-797 (1995)); E. FIG. Kohl et al. (Proc. Nat.
Acad. Sci. U. S. A. 91: 9141-9145 (1994)).

[0358] The oncogene by transformed rodent human Ha-ras or Ki-ras that was mutated (10 ⁶ cells / animal in DMEM salts 1 ml), at day 0 8-1
Inject subcutaneously into the left flank of 2-week-old female nude mice (manufactured by Harlan). Mice in each tumor gene group are randomly assigned to vehicle, compound or combination treatment groups. Animals are dosed subcutaneously on day 1 and are dosed daily for the duration of the experiment. Further, the farnesyl protein transferase inhibitor can be administered by a continuous infusion pump. The compound, compound combination or vehicle is dispensed in a total volume of 0.1 ml. When all vehicle-treated animals show lesions 0.5-1.0 cm in diameter, tumors are excised and weighed, typically 11-15 days after cell injection. The average tumor weight in each treatment group is calculated for each cell group.

[Sequence list]

[Brief description of the drawings]

FIG. 1 is a schematic diagram of the MAPK signal transduction pathway and the activation of the transcription factor Elk1, which leads to the activation of the fos promoter / reporter construct and the expression of secreted alkaline phosphatase (SEAP).

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 31/495 A61K 31/495 4C081 31/496 31/496 4C086 31/5513 31/5513 38/00 A61P 35 / 00 A61P 35/00 43/00 43/00 C12N 9/10 C12N 9/10 C12Q 1/68 Z 15/09 ZNA G01N 33/15 Z C12Q 1/68 33/50 Z G01N 33/15 C07D 233/64 106 33/50 233/90 // C07D 233/64 106 241/04 233/90 401/06 241/04 401/12 401/06 403/06 401/12 403/14 403/06 A61K 37/02 403 / 14 C12N 15/00 ZNAA (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE) , OA (BF, BJ, CF, CG, CI CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY) , KG, KZ, MD, RU, TJ, TM), AL, AM, AU, AZ, BA, BB, BG, BR, BY, CA, CN, CU, CZ, EE, GE, HR, HU, ID , IL, IS, JP, KG, KR, KZ, LC, LK, LR, LT, LV, MD, MG, MK, MN, MX, NO, NZ, PL, RO, RU, SG, SI, SK, SL, TJ, TM, TR, TT, UA, US, UZ, VN, YU (72) Inventor Defo-Jones, Deborah United States of America, New Jersey 07065, Lowway, East Lincoln Avenue 126 (72) Inventor Olif, Allen-I-America United States, New Jersey 07065, Lowway, East Lincoln Ave. 126 (72) Inventor Star Dante, Steven M. United States, New Jersey 07065, Lowway, East Lincoln Ave. 126F term (reference) 2G045 AA26 AA40 BB20 CB01 CB02 DA36 DA78 FB01 4B024 AA11 BA11 BA80 CA02 DA03 EA04 GA11 GA18 HA11 4B050 CC03 CC10 DD11 LL03 4B063 QA01 QA05 QQ08 QQ13 QQ33 QQ43 QQ44 QQ61 QQ95 QR33 QR60 QR62 QR77 QR80 QS24 QS25 QX02 4C063 AA01 AA03 BB03 BB09 CC23 CC34 CC37 CC76 DD03 DD10 DD12 DD23 EE01 4C084 AA18 NA14 ZB262 4C086 AA01 AA02 BC38 BC50 BC56 GA07 GA08 GA12 MA01 MA04 NA14 ZB26

Claims (79)

[Claims] 1. Inhibiting prenyl protein transferase comprising administering a pharmaceutically effective amount of a compound that is a dual inhibitor of farnesyl protein transferase and geranylgeranyl protein transferase type I to a mammal in need thereof. The method, wherein the compound comprises: a) 12 μL for K4B-Ras dependent activation of MAP kinase in cells.
Low inhibitory activity than M _{(IC 50),} and b) K4B-Ras of the MAP kinase at low cell 5 times more than the inhibitory activity against Myr-Ras dependent activation of MAP kinase in cells _{(IC 50)}
A method characterized by an inhibitory activity (IC ₅₀ ) on dependent activation. 2. The compound according to claim 1, wherein the compound further comprises: c) an inhibitory activity (IC ₅₀ ) of less than 10 nM on H-Ras-dependent activation of MAP kinase in cells. The method described in. 3. The MAP kinases K4B-Ras and Myr- in cells.
The inhibitory activity (IC ₅₀ ) of the compound on Ras-dependent activation is determined by: a) Co-transfecting cells with an expression plasmid for the ras gene and an expression plasmid for the reporter construct encoding the product of the reporter gene. 2. The method of claim 1, wherein the assay comprises the steps of: b) incubating the cells in the presence of the test compound; and c) analyzing the lysate of the assay medium or cells for the presence of the reporter gene product. 4. The method according to claim 3, wherein the product of the reporter gene is secreted alkaline phosphatase. 5. The compound further characterized by: c) an inhibitory activity (IC ₅₀ ) of less than 10 nM on H-Ras dependent activation of MAP kinase in the cell, the K4b-Ras and Myr-Ras-dependent inhibitory activity of the compounds on the activation _{(IC 50)} determined by the assay method according to claim 3. 6. The method according to claim 3, wherein the cell is a C33a cell. 7. The method according to claim 4, wherein the cell is a C33a cell. 8. The method according to claim 3, wherein the expression of the reporter gene is controlled by a transcription factor activated by MAP kinase. 9. The method of claim 4, wherein the expression plasmid for the reporter construct encoding secreted alkaline phosphatase is the pDSE101 plasmid. 10. The expression plasmid for a reporter construct encoding secreted alkaline phosphatase is the pDSE101 plasmid.
The method described in. 11. The expression plasmid for the K4B-Ras gene is pSMS.
The method according to claim 4, which is a 640 plasmid or a pZip-rasK4B plasmid. 12. The expression plasmid for the Myr-Ras gene is pSMS.
The method according to claim 5, which is a 600 plasmid. 13. A prenyl protein transferase inhibitor comprising administering to a mammal in need thereof a pharmaceutically effective amount of a compound that is a dual inhibitor of farnesyl protein transferase and geranylgeranyl protein transferase type I. The method, wherein the compound comprises: a) 12 μL for K4B-Ras dependent activation of MAP kinase in cells.
Low inhibitory activity than M _{(IC 50),} and b) less 5 times more than constitutively inhibit activity pCMV-SEAP plasmid expressing SEAP protein on the expression of SEAP protein in the transfected cells in _{(IC 50)} A method characterized by its inhibitory activity (IC ₅₀ ) on K4B-Ras-dependent activation of MAP kinase in cells. 14. Inhibiting prenyl protein transferase comprising administering to a mammal in need thereof a pharmaceutically effective amount of a compound that is a dual inhibitor of farnesyl protein transferase and geranylgeranyl protein transferase type I. The method wherein the compound comprises: a) more than 2 times lower but not more than 20000 times lower inhibitory activity (IC ₅₀ ) on H-ras-CVLL (SEQ ID NO: 1) dependent activation of MPA kinase in cells. Inhibitory activity on H-Ras dependent activation of MAP kinase in cells (I
C _50); and b) constitutively MAP kinase of pCMV-SEAP plasmid expressing SEAP protein at low cell 5 times or more inhibition on the expression of SEAP protein in the transfected cells in the active _{(IC 50)} A method characterized by the inhibitory activity (IC ₅₀ ) of H. ras-CVLL-dependent activation of A. 15. Inhibiting prenyl protein transferase comprising administering to a mammal in need thereof a pharmaceutically effective amount of a compound that is a dual inhibitor of farnesyl protein transferase and geranylgeranyl protein transferase type I. The method wherein the compound comprises: a) more than 2 times lower but not more than 20000 times lower inhibitory activity (IC ₅₀ ) on H-ras-CVLL (SEQ ID NO: 1) dependent activation of MPA kinase in cells. Inhibitory activity on H-Ras dependent activation of MAP kinase in cells (I
C ₅₀ ); and b) H-ras-C of MAP kinase in cells that is more than 5 times lower than the inhibitory activity (IC ₅₀ ) on Myr-Ras dependent activation of MAP kinase in cells.
A method characterized by its inhibitory activity (IC ₅₀ ) on VLL-dependent activation. 16. A farnesyl protein transferase and geranylgeranyl protein comprising administering to a mammal in need thereof a pharmaceutically effective amount of a compound that is a dual inhibitor of farnesyl protein transferase and geranylgeranyl protein transferase type I. A method for inhibiting transferase type I, wherein the compound comprises: a) 12 μm for K4B-Ras dependent activation of MPA kinase in cells.
M) inhibitory activity (IC ₅₀ ) lower than M; and b) K4B-Ras of MAP kinase in cells more than 5-fold lower than the inhibitory activity (IC ₅₀ ) on Myr-Ras dependent activation of MAP kinase in cells.
A method characterized by an inhibitory activity (IC ₅₀ ) on dependent activation. 17. The compound according to claim 16, wherein said compound is further characterized by: c) an inhibitory activity (IC ₅₀ ) of less than 10 nM against H-Ras dependent activation of MAP kinase in cells. The method described in. 18. The MAP kinase K4B-Ras and Myr in cells
The inhibitory activity of compounds against -Ras dependent activation of (IC _{50): a)} the expression plasmid for reporter construct encoding a product of the expression plasmid and a reporter gene for ras gene co-transfected into cells 17. The method of claim 16, wherein the assay comprises determining the presence of a reporter gene product by assaying the assay medium or cell lysate for the presence of a reporter gene product. 19. The method according to claim 18, wherein the product of the reporter gene is secreted alkaline phosphatase. 20. The compound, further characterized by: c) an inhibitory activity (IC ₅₀ ) of less than 10 nM on H-Ras dependent activation of MAP kinase in the cell; the K4b-Ras and Myr-Ras-dependent inhibitory activity of the compounds on the activation _{(IC 50)} determined by the assay method according to claim 18. 21. The method according to claim 18, wherein the cell is a C33a cell. 22. The method according to claim 19, wherein the cell is a C33a cell. 23. The method according to claim 19, wherein the expression of the reporter gene is controlled by a transcription factor activated by MAP kinase. 24. The expression plasmid for a reporter construct encoding secreted alkaline phosphatase is the pDSE101 plasmid.
10. The method according to 9. 25. The expression plasmid for a reporter construct encoding secreted alkaline phosphatase is the pDSE101 plasmid.
3. The method according to 3. 26. The expression plasmid for the K4B-ras gene is pSMS.
The method according to claim 18, which is a 640 plasmid or a pZip-rasK4B plasmid. 27. The expression plasmid for the Myr-ras gene is pSMS.
19. The method according to claim 18, which is a 600 plasmid. 28. A method of treating cancer comprising administering to a mammal in need thereof a pharmaceutically effective amount of a compound that is a dual inhibitor of farnesyl protein transferase and geranylgeranyl protein transferase type I. Thus, the compound comprises: a) 12 μl for K4B-Ras dependent activation of MAP kinase in cells
Low inhibitory activity than M _{(IC 50),} and b) K4B-Ras of the MAP kinase at low cell 5 times more than the inhibitory activity against Myr-Ras dependent activation of MAP kinase in cells _{(IC 50)}
A method characterized by an inhibitory activity (IC ₅₀ ) on dependent activation. 29. The compound according to claim 28, wherein the compound is further characterized by: c) an inhibitory activity (IC ₅₀ ) of less than 10 nM against H-Ras dependent activation of MAP kinase in cells. The method described in. 30. MAP kinase K4B-Ras and Myr in cells
The inhibitory activity of compounds against -Ras dependent activation of (IC _{50): a)} the expression plasmid for reporter construct encoding a product of the expression plasmid and a reporter gene for ras gene co-transfected into cells 29. The method of claim 28, wherein the assay comprises the steps of: b) incubating the cells in the presence of the test compound; and c) analyzing the lysate of the assay medium or cells for the presence of the reporter gene product. 31. The method according to claim 30, wherein the product of the reporter gene is secreted alkaline phosphatase. 32. The method of claim 30, wherein said compound further comprises: c) an inhibitory activity (IC ₅₀ ) of less than 10 nM on H-Ras dependent activation of MAP kinase in cells. . 33. The method according to claim 30, wherein the cells are C33a cells. 34. The method according to claim 31, wherein the cells are C33a cells. 35. The method according to claim 30, wherein the expression of the reporter gene is controlled by a transcription factor activated by MAP kinase. 36. The expression plasmid for a reporter construct encoding secreted alkaline phosphatase is the pDSE101 plasmid.
2. The method according to 1. 37. The expression plasmid for a reporter construct encoding secreted alkaline phosphatase is the pDSE101 plasmid.
5. The method according to 5. 38. The expression plasmid for the K4B-Ras gene is pSMS.
31. The method according to claim 30, which is a 640 plasmid or a pZip-rasK4B plasmid. 39. The expression plasmid for the Myr-Ras gene is pSMS.
31. The method according to claim 30, which is a 640 plasmid or a pSMS621 plasmid. 40. The method of claim 28, wherein the cancer is a cancer characterized by a mutated K4B-Ras gene. 41. A method of treating cancer comprising administering to a mammal in need thereof a pharmaceutically effective amount of a compound that is a dual inhibitor of farnesyl protein transferase and geranylgeranyl protein transferase type I. Thus, the compound comprises: a) 12 μl for K4B-Ras dependent activation of MAP kinase in cells
B) Inhibitory activity (IC ₅₀ ) lower than M, and b) Cells more than 5 times lower than the inhibitory activity (IC ₅₀ ) on expression of SEAP protein in cells transfected with pCMV-SEAP plasmid constitutively expressing SEAP protein A method characterized by the inhibitory activity (IC ₅₀ ) of MAP kinase on K4B-Ras-dependent activation in E. coli. 42. A method for treating cancer comprising administering to a mammal in need thereof a pharmaceutically effective amount of a compound that is a dual inhibitor of farnesyl protein transferase and geranylgeranyl protein transferase type I. Wherein the compound is a) a cell that is at least two times lower but at most 20,000 times lower than the inhibitory activity (IC ₅₀ ) on H-ras-CVLL (SEQ ID NO: 1) dependent activation of MPA kinase in the cell. Activity against H-Ras-dependent activation of MAP kinase in yeast (I
C _50); and b) constitutively MAP kinase of pCMV-SEAP plasmid expressing SEAP protein at low cell 5 times or more inhibition on the expression of SEAP protein in the transfected cells in the active _{(IC 50)} A method characterized by the inhibitory activity (IC ₅₀ ) of H. ras-CVLL-dependent activation of A. 43. A method for treating cancer comprising administering to a mammal in need thereof a pharmaceutically effective amount of a compound that is a dual inhibitor of farnesyl protein transferase and geranylgeranyl protein transferase type I. Wherein the compound is a) a cell that is at least two times lower but at most 20,000 times lower than the inhibitory activity (IC ₅₀ ) on H-ras-CVLL (SEQ ID NO: 1) dependent activation of MPA kinase in the cell. Activity against H-Ras-dependent activation of MAP kinase in yeast (I
C ₅₀ ); and b) H-ras-C of MAP kinase in cells that is more than 5 times lower than the inhibitory activity (IC ₅₀ ) on Myr-Ras dependent activation of MAP kinase in cells.
A method characterized by its inhibitory activity (IC ₅₀ ) on VLL-dependent activation. 44. The method of claim 28, wherein said compound is selected from the following compounds or pharmaceutically acceptable salts thereof: 1- [2 (R) -amino-3-mercaptopropyl] -2 ( S)-[(3-Pyridyl) methoxyethyl)]-4- (1-naphthoyl) piperazine 1- [2 (R) -amino-3-mercaptopropyl] -2 (S)-(benzyloxymethyl) -4 -(1-Naphthoyl) piperazine 1- [2 (R) -amino-3-mercaptopropyl] -2 (S)-(benzyloxymethyl) -4- [7- (2,3-dihydrobenzofuroyl)] Piperazine 1- [2 (R) -amino-3-mercaptopropyl] -2 (S)-(benzamido) -4- (1-naphthoyl) piperazine 1- [2 (R) -amino-3-mercaptopropyl]- 2 (S) [4- (5
-Dimethylamino-1-naphthalenesulfonamido) -1-butyl] -4- (1
-Naphthoyl) piperazine N- [2 (S)-(1- (4-nitrophenylmethyl) -1H-imidazol-5-ylacetyl) amino-3 (S) -methylphenyl] -N-1-naphthylmethyl-glycyl -Methionine N- [2 (S)-(1- (4-nitrophenylmethyl) -1H-imidazol-5-ylacetyl) amino-3 (S) -methylphenyl] -N-1-naphthylmethyl-glycyl-methionine Methyl ester N- [2 (S)-[(1- (4-cyanobenzyl) -1H-imidazole-5
-Yl] acetylamino) -3 (S) -methylpentyl] -N- (1-naphthylmethyl) glycyl-methionine N- [2 (S)-[(1- (4-cyanobenzyl) -1H-imidazole- 5
-Yl] acetylamino) -3 (S) -methylpentyl] -N- (1-naphthylmethyl) glycyl-methionine methyl ester 2 (S) -n-butyl-4- (1-naphthoyl) -1- [1 -(2-naphthylmethyl) imidazol-5-ylmethyl] -piperazine 2 (S) -n-butyl-1-[(1- (4-cyanobenzyl) imidazole-
5-ylmethyl] -4- (1-naphthoyl) piperazine 1-{[1- (4-cyanobenzyl) -1H-imidazol-5-yl] acetyl} -2 (S) -n-butyl-4- (1 -Naphthoyl) piperazine 1- (3-chlorophenyl) -4- [1- (4-cyanobenzyl) imidazolylmethyl] -2-piperazinone 1-phenyl-4- [1- (4-cyanobenzyl) -1H-imidazole-5
-Ylethyl] -piperazin-2-one 1- (3-trifluoromethylphenyl) -4- [1- (4-cyanobenzyl) -1H-imidazol-5-ylmethyl] -piperazin-2-one 1- (3- Bromophenyl) -4- [1- (4-cyanobenzyl) -1H-imidazol-5-ylmethyl] -piperazin-2-one 5 (S) -2- [2,2,2-trifluoroethoxy] ethyl)- 1- (3-
Trifluoromethylphenyl) -4- [1- (4-cyanobenzyl) -4-imidazolylmethyl] -piperazin-2-one 1- (5,6,7,8-tetrahydronaphthyl) -4- [1- ( 4-cyanobenzyl) -1H-imidazol-5-ylmethyl] -piperazin-2-one 1- (2-methyl-3-chlorophenyl) -4- [1- (4-cyanobenzyl) -4-imidazolylmethyl]- Piperazin-2-one 2 (RS)-{[1- (naphth-2-ylmethyl) -1H-imidazole-5
-Yl) acetyl {amino-3- (t-butoxycarbonyl) amino-N- (2
-Methylbenzyl) propionamide N- {1- (4-cyanobenzyl) -1H-imidazol-5-ylmethyl}
-4 (R) -benzyloxy-2 (S)-{N'-acetyl-N'-3-chlorobenzyl} aminomethylpyrrolidine N- {1- (4-cyanobenzyl) -1H-imidazol-5-ylethyl}
-4 (R) -benzyloxy-2 (S)-{N'-acetyl-N'-3-chlorobenzyl} aminomethylpyrrolidine 1- [1- (4-cyanobenzyl) -1H-imidazol-5-ylacetyl} Pyrrolidin-2 (S) -ylmethyl]-(N-2-methylbenzyl) -glycine N '-(3-chlorophenylmethyl) amide 1- [1- (4-cyanobenzyl) -1H-imidazol-5-ylacetyl} Pyrrolidin-2 (S) -ylmethyl]-(N-2-methylbenzyl) -glycine N′-methyl-N ′-(3-chlorophenylmethyl) amide (S) -2-[(1- (4-cyanobenzyl) ) -5-Imidazolylmethyl) amino] -N- (benzyloxycarbonyl) -N- (3-chlorobenzyl) -4-
(Methanesulfonyl) butanamine 1- (3,5-dichlorobenzenesulfonyl) -3 (S)-[N- (1- (4
-Cyanobenzyl) -1H-imidazol-5-ylmethyl) carbamoyl] piperidine N-{[1- (4-cyanobenzyl) -1H-imidazol-5-yl] methyl} -4- (3-methylphenyl) -4 -Hydroxypiperidine N-{[1- (4-cyanobenzyl) -1H-imidazol-5-yl] methyl} -4- (3-chlorophenyl) -4-hydroxypiperidine 4- [1- (4-cyanobenzyl) -5-imidazolylmethyl] -1- (2,
3-dimethylphenyl) -piperazine-2,3-dione 1- (2- (3-trifluoromethoxyphenyl) -pyrid-5-ylmethyl) -5- (4-cyanobenzyl) imidazole 4- {5- [1 -(3-chloro-phenyl) -2-oxo-1,2-dihydro-pyridin-4-ylmethyl] -imidazol-1-ylmethyl} -2-methoxy-benzonitrile 3 (R) -3- [1- ( 4-cyanobenzyl) imidazol-5-yl-ethylamino] -5-phenyl-1- (2,2,2-trifluoroethyl) -H-benzo [e] [1,4] diazepine 3 (S)- 3- [1- (4-cyanobenzyl) imidazol-5-yl-ethylamino] -5-phenyl-1- (2,2,2-trifluoroethyl) -H-benzo [e] [1,4] Diazepi N- {1- (4-cyanobenzyl) -1H-imidazol-5-ylacetyl) pyrrolidin-2 (S) -ylmethyl] -N- (1-naphthylmethyl) glycyl-methionine N- {1- (4- Cyanobenzyl) -1H-imidazol-5-ylacetyl) pyrrolidin-2 (S) -ylmethyl] -N- (1-naphthylmethyl) glycyl-methionine methyl ester N- [1- (1H-imidazol-4-ylpropionyl) Pyrrolidine-2 (
S) -ylmethyl] -N- (2-methoxybenzyl) glycyl-methionine N- [1- (1H-imidazol-4-ylpropionyl) pyrrolidine-2 (
S) -ylmethyl] -N- (2-methoxybenzyl) glycyl-methionine methyl ester 2 (S)-(4-acetamido-1-butyl) -1- [2 (R) -amino-3
-Mercaptopropyl] -4- (1-naphthoyl) piperazine 2 (RS)-{[1- (naphth-2-ylmethyl) -1H-imidazole-5
-Yl)] acetyl {amino-3- (t-butoxycarbonyl) amino-N-cyclohexyl-propionamide 1- {2 (R, S)-} [1- (4-cyanobenzyl) -1H-imidazole-5 -Yl)] propanol {-2 (S) -n-butyl-4- (1-naphthoyl) piperazine 1- [1- (4-cyanobenzyl) imidazol-5-ylmethyl] -4- (
Diphenylmethyl) piperazine 1- (diphenylmethyl) -3 (S)-[N- (1- (4-cyanobenzyl)
-2-methyl-1H-imidazol-5-ylmethyl) -N- (acetyl) aminomethyl] piperidine N- [1- (1H-imidazol-4-ylpropionyl) pyrrolidine-2 (
S) -ylmethyl] -N- (2-chlorobenzyl) glycyl-methionine N- [1- (1H-imidazol-4-ylpropionyl) pyrrolidine-2 (
S) -ylmethyl] -N- (2-chlorobenzyl) glycyl-methionine methyl ester 3 (R) -3- [1- (4-cyanobenzyl) imidazol-5-yl-methylamino] -5-phenyl-1- (2,2,2-trifluoroethyl) -H-benzo [e] [1,4] diazepine 1- (3-trifluoromethoxyphenyl) -4- [1- (4-cyanobenzyl) imidazolylmethyl]- 2-piperazinone 1- (2,5-dimethylphenyl) -4- [1- (4-cyanobenzyl) imidazolylmethyl] -2-piperazinone 1- (3-methylphenyl) -4- [1- (4-cyano Benzyl) imidazolylmethyl] -2-piperazinone 1- (3-iodophenyl) -4- [1- (4-cyanobenzyl) imidazolylmethyl] -2-piperazino 1- (3-chlorophenyl) -4- [1- (4-cyano-3-methoxybenzyl) imidazolylmethyl] -2-piperazinone 1- (3-trifluoromethoxyphenyl) -4- [1- (4-cyano -3-
Methoxybenzyl) imidazolylmethyl] -2-piperazinone 4-[((1- (4-cyanobenzyl) -5-imidazolyl) methyl) amino] benzophenone 1- (1-{[3- (4-cyanobenzyl) -3H] -Imidazol-4-yl] -acetyl} -pyrrolidin-2 (S) -ylmethyl) -3 (S) -ethyl-pyrrolidin-2 (S) -carboxylic acid 3-chloro-benzylamide. 45. A method of inhibiting prenyl protein transferase comprising administering a pharmaceutically effective amount of a compound to a mammal in need thereof, said compound comprising: a) MAP kinase in a cell. 12 μl for K4B-Ras dependent activation of
A method characterized by an inhibitory activity (IC ₅₀ ) lower than M. 46. An assay for identifying a prenyl protein transferase inhibitor that is effective in vivo as an inhibitor of the biological activity of Ras protein, comprising: a) an expression plasmid for the ras gene and a reporter gene. Co-transfecting the cells with an expression plasmid for a reporter construct encoding the product of b) incubating the cells in the presence of the test compound, and c) assaying for the presence of the product of the reporter gene or Assay comprising analyzing cell lysates. 47. The assay according to claim 46, wherein the product of the reporter gene is secreted alkaline phosphatase. 48. The assay according to claim 46, wherein the cells are C33a cells. 49. The expression plasmid for a reporter construct encoding secreted alkaline phosphatase is the pDSE101 plasmid.
7. The assay according to 7. 50. The assay according to claim 46, wherein the Ras protein is K4B-Ras. 51. The assay of claim 50, wherein the prenyl protein transferase inhibitor is a dual inhibitor of farnesyl protein transferase and geranylgeranyl protein transferase type I. 52. The expression plasmid for a reporter construct encoding secreted alkaline phosphatase is the pDSE101 plasmid.
Assay according to 0. 53. A method for identifying a prenyl protein transferase inhibitor that is effective in vivo as an inhibitor of cancer cell growth, comprising: a) the ras gene is K-ras. B) evaluating the test compound in the assay of claim 46, wherein the ras gene is Myr-Ras; and c) Myr-Ras dependent activation of MAP kinase in the assay of claim 46. Comparing the activity of a test compound to K-Ras-dependent activation of MAP kinase in the assay of claim 46. 54. The method according to claim 53, wherein the K-ras gene is K4B-ras. 55. The method according to claim 54, wherein the prenyl protein transferase inhibitor is a dual inhibitor of farnesyl protein transferase and geranylgeranyl protein transferase type I. 56) cotransfecting the cells with an expression plasmid for the ras gene and an expression plasmid for a reporter construct encoding the product of the reporter gene; b) transforming the cells in the presence of the test compound Incubating, and c) analyzing the lysate of the assay medium or cells for the presence of the product of the reporter gene. D) assessing the test compound in an assay comprising: 55. The method of claim 54, wherein -ras. 57. The method according to claim 56, wherein the product of the reporter gene is secreted alkaline phosphatase. 58. a) co-transfecting the cells with an expression plasmid for the ras gene and an expression plasmid for the reporter construct encoding the product of the reporter gene; b) transforming the cells in the presence of the test compound Incubating, and c) analyzing the lysate of the assay medium or cells for the presence of the product of the reporter gene. D) assessing the test compound in an assay comprising: 55. The method of claim 54, wherein the method is -ras-CVLL. 59. The method according to claim 58, wherein the product of the reporter gene is secreted alkaline phosphatase. 60) a) co-transfecting cells with an expression plasmid for the ras gene and an expression plasmid for a reporter construct encoding the product of the reporter gene; b) transforming the cells in the presence of the test compound Incubating, and c) analyzing the lysate of the assay medium or cells for the presence of the product of the reporter gene. E) evaluating the test compound in an assay comprising: 57. The method of claim 56, wherein the method is -ras-CVLL. 61. The method according to claim 60, wherein the product of the reporter gene is secreted alkaline phosphatase. 62. A method for identifying a prenyl protein transferase inhibitor that is effective in vivo as an inhibitor of cancer cell growth, comprising: a) the ras gene is H-Ras. Evaluating the test compound in b) evaluating the test compound in the assay of claim 46, wherein the ras gene is H-ras-CVLL; c) testing in the assay of claim 46, wherein the ras gene is Myr-ras. Evaluating the compound; and d) determining the activity of the test compound on the Myr-Ras dependent activation of MAP kinase in the assay of claim 46, and the H-Ras dependent activation of MAP kinase in the assay of claim 46. H-ras-CVLL-dependent activity of MAP kinase in the assay of paragraph 46 Comprising the step of comparing the activity of test compounds against. 63. The method according to claim 62, wherein the product of the reporter gene is secreted alkaline phosphatase. 64. The method of claim 62, wherein the prenyl protein transferase inhibitor is a dual inhibitor of farnesyl protein transferase and geranylgeranyl protein transferase type I. 65. A method for identifying a prenyl protein transferase inhibitor that is effective in vivo as an inhibitor of cancer cell growth, comprising: a) the ras gene is N-Ras. B) evaluating the test compound in the assay of claim 46, wherein the ras gene is Myr-ras; and c) Myr-Ras dependent activation of MAP kinase in the assay of claim 46. Comparing the activity of a test compound to N-Ras-dependent activation of MAP kinase in the assay of claim 46. 66. The method according to claim 65, wherein the product of the reporter gene is secreted alkaline phosphatase. 67. A method for identifying a prenyl protein transferase inhibitor that is effective in vivo as an inhibitor of cancer cell growth, comprising: a) the ras gene is H-Ras. Evaluating the test compound in b) evaluating the test compound in the assay of claim 46, wherein the ras gene is H-ras-CVLL; c) testing in the assay of claim 46, wherein the ras gene is Myr-ras. Assessing the compound; and d) measuring the activity of the test compound on H-ras-CVLL-dependent activation of MAP kinase in the assay of claim 46.
Comparing the activity of the test compound to the H-Ras dependent activation of the kinase with the activity of the test compound; e) comparing the activity of the test compound to the Myr-Ras dependent activation of the MAP kinase in the assay of claim 46; Comparing the activity of the test compound to H-ras-CVLL-dependent activation of MAP kinase. 68. A method for identifying a prenyl protein transferase inhibitor that is effective in vivo as an inhibitor of cancer cell growth, comprising: a) the ras gene is K-Ras. B) evaluating the test compound in an assay comprising the following steps a) to c): a) transfecting the cells with the pCMV-SEAP plasmid, b) the presence of the test compound Incubating the cells underneath, and c) analyzing the lysate of the assay medium or cells for the presence of the product of the reporter gene; c) providing a test compound for expression of SEAP protein in cells transfected with the pCMV-SEAP plasmid. 47. The activity of the MAP in the assay of claim 46.
Comparing the activity of the test compound to K-ras dependent activation of the kinase. 69. A method for identifying a prenyl protein transferase inhibitor that is effective in vivo as an inhibitor of cancer cell growth, comprising: a) the ras gene is H-Ras. B) evaluating the test compound in the assay of claim 46, wherein the ras gene is H-ras-CVLL; c) testing the test compound in an assay comprising the following steps a) to c): Assessing: a) transfecting the cells with the pCMV-SEAP plasmid, b) incubating the cells in the presence of the test compound, and c) lysate of the assay medium or cells for the presence of the product of the reporter gene. Analyzing the MAP kinase H-ras-C in the assay of claim 46. The activity of test compounds against LL-dependent activation, MAP in the assay of claim 46
Comparing the activity of the test compound to the H-ras dependent activation of the kinase with the activity of the test compound; e) testing the activity of the test compound for expression of the SEAP protein in cells transfected with the pCMV-SEAP plasmid;
Comparing the activity of the test compound to H-ras-CVLL-dependent activation of the kinase. 70. The method of claim 65, wherein the prenyl protein transferase inhibitor is a dual inhibitor of farnesyl protein transferase and geranylgeranyl protein transferase type I. 71. A method for identifying a combination of a selective inhibitor of geranylgeranyl protein transferase type I and a farnesyl protein transferase that is effective in vivo as an inhibitor of cancer cell growth in vivo. Wherein: a) the ras gene is selected from K4B-ras and N-ras.
Evaluating a test combination of a selective inhibitor of geranylgeranyl protein transferase type I and a farnesyl protein transferase in the assay of 6; b) the ras gene is Myr-ras in the assay of 46. Evaluating a test combination of a selective inhibitor of protein transferase type I and a selective inhibitor of farnesyl protein transferase;
And c) the activity of a test combination of a selective inhibitor of geranylgeranyl protein transferase type I and a farnesyl protein transferase on Myr-Ras dependent activation of MAP kinase in the assay of claim 46. Activity of test combinations of a selective inhibitor of geranylgeranyl protein transferase type I and a farnesyl protein transferase on the dependent activation of MAP kinase by the protein encoded by the gene of step a) in the assay of 46 Comparing with the method. 72. The method according to claim 71, wherein the product of the reporter gene is secreted alkaline phosphatase. 73. The method according to claim 71, wherein the ras gene is K4B-ras. 74. The method according to claim 71, wherein the ras gene is N-ras. 75. d) A further step of evaluating a test combination of a selective inhibitor of geranylgeranyl protein transferase type I and a farnesyl protein transferase in the assay of claim 46, wherein the ras gene is H-ras. 72. The method of claim 71, further comprising: 76. d) The ras gene is H-ras-CVLL.
73. The method of claim 71, further comprising the step of evaluating a test combination of a selective inhibitor of geranylgeranyl protein transferase type I and a farnesyl protein transferase in the assay of 6. 77. d) A further step of evaluating a test combination of a selective inhibitor of geranylgeranyl protein transferase type I and a farnesyl protein transferase in the assay of claim 46, wherein the ras gene is H-ras. And e) evaluating the test combination of a selective inhibitor of geranylgeranyl protein transferase type I and a farnesyl protein transferase in the assay of claim 46, wherein the ras gene is H-ras-CVLL. 72. The method of claim 71, further comprising: 78. a) evaluating the test combination in the assay of claim 46, wherein the ras gene is K-Ras; b) evaluating the test combination in an assay comprising the following steps a) -c): Steps: a) transfecting the cells with the pCMV-SEAP plasmid, b) incubating the cells in the presence of the test combination, and c) analyzing the lysate of the assay medium or cells for the presence of the product of the reporter gene. C) measuring the activity of the test combination on expression of SEAP protein in cells transfected with the pCMV-SEAP plasmid by M in the assay of claim 46.
47. A test combination of a selective inhibitor of geranylgeranyl protein transferase type I with a selective inhibitor of farnesyl protein transferase in the assay of claim 46 by comparing the activity of the test combination to K-ras dependent activation of AP kinase. Identifying a combination of a selective inhibitor of geranylgeranyl protein transferase type I and a farnesyl protein transferase that is effective in vivo as an inhibitor of cancer cell growth, comprising evaluating the product Way to do. 79. a) evaluating the test combination in the assay of claim 46, wherein the ras gene is H-Ras; b) testing the test combination in the assay of claim 46, wherein the ras gene is H-ras-CVLL. C) evaluating the test combination in an assay comprising the following steps a) to c): a) transfecting the cells with the pCMV-SEAP plasmid, b) in the presence of the test combination Incubating the cells in the assay medium or c) analyzing the lysate of the assay medium or cells for the presence of the product of the reporter gene; d) inhibiting H-ras-CVLL-dependent activation of MAP kinase in the assay of claim 46. The activity of the test combination is determined by measuring the H of MAP kinase in the assay of claim 46. Comparing the activity of the test combination to the expression of the SEAP protein in cells transfected with the pCMV-SEAP plasmid by comparing the activity of the test combination to cells transfected with the pCMV-SEAP plasmid; MAP kinase in the assay of claim 46. 47. A test combination of a selective inhibitor of geranylgeranyl protein transferase type I and a selective inhibitor of farnesyl protein transferase in the assay of claim 46 by comparing the activity of the test combination to H-Ras-CVLL-dependent activation of A selective inhibitor of geranylgeranyl protein transferase type I and a farnesyl protein transferase that is effective in vivo as an inhibitor of cancer cell growth comprising assessing Method for identifying only combined product.