Cycloalkyl inhibitors of protein farnesyltransferase

专利摘要:
The present invention describes novel protein farnesyltransferase inhibitors, methods for their preparation and pharmaceutical compositions, useful for modulating tissue proliferative diseases including cancer, restenosis, atherosclerosis, psoriasis and endometriosis.
公开号:KR20000057601A
申请号:KR1019990705384
申请日:1997-12-16
公开日:2000-09-25
发明作者:아네트 마리안 도허티；제임스 스탠리 칼텐브론；다니엘 엠. 레오나르드；데니스 죠셉 맥나마라
申请人:로즈 암스트롱, 크리스틴 에이. 트러트웨인；워너-램버트 캄파니；
IPC主号:

专利说明:

Cycloalkyl Inhibitors of Protein Farnesyltransferase
The present invention relates to compounds that can be used in the pharmaceutical art for preventing or otherwise treating uncontrolled or abnormal tissue proliferation. Specifically, the present invention relates to compounds that activate cell division and inhibit Panesyltransferase, which is designated to activate Ras proteins involved in cancer and restenosis.
Ras protein (or p21) has been extensively studied because its mutant form is found in 20% of most types of human cancer and in more than 50% of colon and pancreatic cancers (Gibbs JB). Cell, 1991; 65: 1, Cartwright T. et al., Chimica. Oggi., 1992; 10: 26). These mutant Ras proteins lack the feedback regulators present in wild Las, and their lack of oncogenic action is not possible because the ability to stimulate normal cell division cannot be controlled by normal endogenous regulatory cofactors. Is associated with. Recent findings that the transgenic activity of mutant Ras are critically dependent on post-translational mutations (Gibbs J. et al. Microbiol. Rev., 1989; 53: 171) are important for Ras function. It revealed a new face and revealed new prospects for cancer treatment.
In addition to cancer, there are other uncontrolled cell proliferation states that may have been associated with overexpression and / or function of wild Ras proteins. Vascular restenosis after surgery is such a condition. The use of various surgical revascularization techniques, such as saphenous vein arthroplasty, endometrial resection, and percutaneous coronary angioplasty, is often associated with uncontrolled growth of neointimal tissue, known as restenosis. Entails. The biochemical causes of restenosis are not well understood and many growth factors and proto-tumor genes are involved (Naftilan AJ, et al., Hypertension, 1989; 13: 706) and literature [ J. Clin. Invest., 83: 1419; Gibbons GH et al., Hypertension, 1989; 14: 358; Satoh T. et al., Molec. Cell Biol., 1993; 13: 3706]. The fact that Ras proteins are known to be involved in the process of cell division can be suspected as intervening in many situations in which cells divide and divide. By direct analogy to the inhibition of mutant Ras-related cancers, blockade of the Ras-dependent process reduces inappropriate tissue proliferation associated with restenosis, especially when normal Ras expression and / or function is amplified by growth stimulating factors or Has the potential to remove
Lars' function depends on the conversion of proteins to bind to the inner surface of the plasma membrane. Unlike other membrane related proteins, Ras proteins lack conventional transmembrane or hydrophobic sequences and are synthesized in a form that is initially soluble in the cytoplasm. Membrane binding of the Ras protein is caused by a series of post-translational processing steps signaled by the carboxyl terminal amino acid consensus sequence recognized by the protein farnesyl transferase (PFT). This consensus sequence consists of a cystine residue located 4 amino acids away from the carboxy terminus, followed by 2 lipophilic amino acids and a C-terminal residue. The sulfhydryl groups of cystine residues are alkylated by faresylpyrophosphate in a reaction in which the protein farnesyltransferase acts as a catalyst. Following prenylation, the three amino acids at the C-terminus are cleaved by the endoprotease and the newly exposed alpha-carboxyl groups of the prenylated cystine are methylated by methyl transferase. Enzymatic treatment of Ras proteins, beginning with farnesylation, allows the proteins to be associated with cell membranes. Mutation analysis of oncogenic Ras proteins shows that these post-translational transformations are important for transformation activity. Replacing cystine residues of the consensus sequence with other amino acids results in a Ras protein that is no longer panicylated, unable to migrate to the cell membrane and lacks the ability to stimulate cell proliferation (Hancock JF et al., Et al. Cell, 1989; 57: 1617, Schafer WR et al., Science, 1989; 245: 379, Casey PJ, Proc. Natl. Acad. Sci. USA, 1989; 86: 8323.
Recently, protein farnesyl transferase (PFT, also called farnesyl protein transferase (FPT)) has been identified and certain PFTs have been purified from the mouse brain to a degree of homogeneity (Reiss Y. et al., Et al. Bioch. Soc. Trans., 1992; 20: 487-88]. The enzyme is characterized by a heterodimer consisting of one alpha-subunit (49kDa) and one beta-subunit (46kDa), both of which require catalytic activity. High concentration expression of mammalian PFTs in the baculovirus system and purification of the active form of recombinant enzymes have also been made (Chen WJ et al., J. Biol. Chem., 1993; 268: 9675). ).
In light of the prior art, the discovery that the function of oncogenic Ras proteins is critically dependent on their post-translational processing provides a means of cancer chemotherapy through the inhibition of processing enzymes. Identification and isolation of protein farnesyltransferases that catalyze the reaction of adding farnesyl groups to the Ras protein provide a promising target for such interference. Ras farnesyl transferase inhibitors have been shown to have anticancer activity in some recent literature.
Lars inhibitor agonists work by inhibiting panesyl transferase, an enzyme that fixes the protein product of the Ras gene to the cell membrane. The role of Ras mutations in the growth signal transduction in cancer cells depends on the protein present in the cell membrane, so if Panesyltransferase is inhibited, the Ras protein will remain in the cytoplasm and cannot transmit growth signals: this fact Is known in the literature.
Peptidedomimetic inhibitors of panesyl transferase B956 and its methyl ester B1086 100 mg / kg have been shown to inhibit tumor growth by human colon cancer xenografts in EJ-1 human bladder cancer, HT1080 human fibrosarcoma and hairless mice ( Nagasu, T., et al., Cancer Res., 1995; 55: 5310-5314. In addition, inhibition of tumor growth by B956 has been shown to correlate with Ras post-translational processing in tumors. Other Ras farnesyltransferase inhibitors have been shown to specifically block Ras treatment and membrane ubiquity and are effective in reversing the transformed phenotype of mutant Ras containing cells (Sepp-Lorenzino L.). Et al., Cancer Res., 1995; 55: 5302-5309.
In another report (Sun J. et al., Cancer Res., 1995; 55: 4243-4247), the Ras farnesyltransferase inhibitor FTI276 is expressed in hairless mice by K-Ras mutation and p53 deletion. Has been shown to selectively prevent tumor growth in human lung cancer. According to another report, the daily dose of las farnesyl transferase inhibitor L-744,832 Administration has resulted in tumor regression of breast and salivary cancers in Las transgenic mice (Kohl et al., Nature Med., 1995; 1 (8): 792-748). Thus, Ras farnesyltransferase inhibitors are beneficial for certain types of cancer, especially cancers whose growth is dependent on tumorigenic Ras. However, it is known that human cancer is often evident when several mutations occur in important genes and that one or more of these mutations may be responsible for the regulation of growth and metastasis. One mutation may not be enough to support growth, and after two of the three mutations, the tumor can develop and grow. Therefore, it is difficult to determine which of these mutations can primarily drive growth in certain types of cancer. Thus, Ras farnesyltransferase inhibitors may have therapeutic utility in tumors that do not rely on growth only on tumorigenic forms of Ras. For example, various Ras farnesyltransferase inhibitors have been shown to have antiproliferative effects in vivo on tumor lines with either wild type or mutant Ras (Sepp-Lorenzino's tactical literature). In addition, there are several las-related proteins that are prenylated. Proteins such as R-ras2 / TC21 are las-related proteins that are prenylated in vivo by both panesyl transferase and geranyl geranyl transferase I (Carboni et al., Oncogene, 1995; 10). 1905-1913]. Thus, Ras farnesyltransferase inhibitors can also prevent prenylation of these proteins and thus be useful for inhibiting tumor growth by other oncogenes.
With regard to restenosis and vasculature, it has been found that inhibition of cellular las prevents smooth muscle proliferation after vascular injury in vivo (Indolfi C et al., Nature Med., 1995; 1 (6)). : 541-545]. This report clearly supports the role of Panesyltransferase inhibitors in this disease, indicating inhibition of the accumulation and proliferation of vascular smooth muscle.
Summary of the invention
The present invention provides compounds of formula (I) and their pharmaceutically acceptable salts, esters, amides and prodrugs thereof.
In the above formula
R ^Q is

ego;
x is 0 or 1;
Each R ¹⁴ is independently hydrogen or C ₁ -C ₆ alkyl;
A is -COR ^a , -CO ₂ R ^{a '} , -CONHR ^a' , -CSR ^a , , , -C (S) OR ^{a '} , -C (S) NHR ^a' , -SO ₂ R ^a or -CONR ^a R ^{a ''} ;
R ^a , R ^{a '} and R ^a'' are independently C ₁ -C ₆ alkyl,-(CR ¹⁴ R ¹⁴ ) _m -cycloalkyl,-(CR ¹⁴ R ¹⁴ ) _m -aryl, or-(CR ¹⁴ R ¹⁴ ) _m -heteroaryl;
Each m is independently 0-3;
R ¹ , R ² and R ⁴ are independently hydrogen or C ₁ -C ₆ alkyl;
R³Is , C_One-C₆Alkyl, C₂-C₆Alkenyl,-(CR¹⁴R¹⁴)_m-Naphthyl,-(CH₂)_vCO₂R¹⁴,-(CH₂)_tNR¹⁴R¹⁴,-(CH₂)_v-O-C_One-C₆Alkyl,-(CH₂)_t-OH,-(CH₂)_tMorpholino,,,-(CR¹⁴R¹⁴)_m-(R^bPhenyl substituted by-or-(CR¹⁴R¹⁴)_m-(R^bHeteroaryl substituted with;
t is 2 to 6;
v is 1 to 6;
R ^b is —O-phenyl, —O-benzyl, halogen, C ₁ -C ₆ alkyl, hydrogen, —OC ₁ -C ₆ alkyl, -NH ₂ , -NHR ^a , -NR ^a R ^{a '} , , , -OH, CF ₃ , -NO ₂ , , , -CN, -OPO ₃ H ₂ , -CH ₂ PO ₃ H ₂ , , , , , , -O (CH ₂ ) _y NR ^a R ^{a '} , -N ₃ , -CF ₂ CF ₃ , -SO ₂ R ^a , -SO ₂ NR ^a R ^a' , -CHO, -OCOCH ₃ , -O (CH ₂ ) _m -heteroaryl, -O (CH ₂ ) _m -aryl, -O (CH ₂ ) _m -cycloalkyl,-(CH ₂ ) _m -aryl,-(CH ₂ ) _m -cycloalkyl,-(CH ₂ ) _m -heteroaryl, or -CH = CHC ₆ H ₅ ;
y is 2 or 3;
R ⁵ is

ego;
Each n is independently 2, 3 or 4;
R ⁱ , R ^g and R ^h are independently hydrogen, halogen, -OC ₁ -C ₆ alkyl, C ₁ -C ₆ alkyl, -CN, -OPO ₃ H ₂ , -CH ₂ PO ₃ H ₂ , -O- Phenyl, -O-benzyl, , , , , -O (CH ₂ ) _y NR ^a R ^{a '} , -NH ₂ , -NHR ^a , -NR ^a R ^a' , , , -OH, CF ₃ , -NO ₂ , , , , -N ₃ , -CF ₂ CF ₃ , -SO ₂ R ^a , -SO ₂ NR ^a R ^{a '} , -CHO or -OCOCH ₃ ;
R ^c and R ^d are independently C ₁ -C ₆ alkyl, — (CH ₂ ) _m -cycloalkyl or hydrogen.
In a preferred embodiment of the compound of formula I
R ¹ is hydrogen, R ² is hydrogen, R ⁴ is hydrogen, R ¹⁴ is hydrogen or methyl,
A is

to be.
In another preferred embodiment of the compound of formula I
R ³ is , C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl,-(CH ₂ ) _m- (phenyl substituted with R ^b ) or-(CH ₂ ) _m- (heteroaryl substituted with R ^b ).
R ¹ is hydrogen, R ² is hydrogen, R ⁴ is hydrogen, and R ¹⁴ is hydrogen or methyl.
In another preferred embodiment of the compound of formula I
R ⁵ is

to be.
The present invention also provides compounds of formula (II) and their pharmaceutically acceptable salts, esters, amides and prodrugs thereof
In the above formula
R ⁶ is -O-benzyl, , -NH-benzyl, -N (C ₁ -C ₆ alkyl) -benzyl or -SCH ₂ -phenyl;
R ⁸ is hydrogen, halogen, C ₁ -C ₆ alkyl, —O-benzyl, —OCH ₂ -pyridyl, —OC ₁ -C ₆ alkyl, —CF ₃ , —OH or —phenyl;
R ¹⁰ and R ¹³ are independently hydrogen or C ₁ -C ₆ alkyl;
Each n is independently 2, 3 or 4;
R ¹² is ego;
R ¹⁴ is hydrogen or methyl;
R^j, R^kAnd R^lIndependently hydrogen, halogen, -NH₂, -NHR^a, -OC_One-C₆Alkyl or -C_One-C₆Alkyl.
The invention also provides compounds of formula III and their pharmaceutically acceptable salts, esters, amides and prodrugs thereof
In the above formula
Each n is 2, 3 or 4;
X is NH, O, or -NCH ₃ ;
R ¹⁵ is —O-benzyl, —CF ₃ , hydrogen, halogen, —OH, —phenyl, —C ₁ -C ₆ alkyl, —OCH ₂ -pyridyl or —OC ₁ -C ₆ alkyl.
The present invention also provides a compound of formula IV
In the above formula
X is NH, O, or -NCH ₃ ;
R ¹⁴ is hydrogen or C ₁ -C ₆ alkyl;
R ^m is C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl,-(CH ₂ ) _t NR ¹⁴ R ¹⁴ ,-(CH ₂ ) _v -OC ₁ -C ₆ alkyl,-(CH ₂ ) _t- OH,-(CH ₂ ) _t -morpholino, , ,-(CH ₂ ) _v -CO ₂ R ¹⁴ or ego;
n is 2, 3 or 4;
m is 0 to 3;
t is 2 to 6;
v is 1-6.
In another aspect, the present invention provides a pharmaceutically acceptable composition comprising a compound of Formula (I), (II), (III) or (IV).
Also provided are methods of treating or preventing restenosis, including administering to a patient with or at risk of restenosis, a therapeutically effective amount of a compound of Formula (I), (II), (III) or (IV).
Also provided are methods of treating cancer, i.e., comprising administering to a patient with cancer a compound of formula (I), (II), (III) or (IV) in a therapeutically effective amount.
In a more preferred embodiment, the cancer is lung cancer, colon cancer, breast cancer, pancreatic cancer, thyroid cancer or bladder cancer.
In a most preferred embodiment, the compound of formula I, II, III or IV,
(S)-[1-((4-benzyloxy-benzyl)-{[(1-phenyl-cyclobutylmethyl) -carbamoyl] -methyl} -carbamoyl) -2- (1H-imidazole-4- Yl) -ethyl] -carbamic acid benzyl ester;
(S)-[1-((4-benzyloxy-benzyl)-{[(1-phenyl-cyclopropylmethyl) -carbamoyl] -methyl} -carbamoyl) -2- (1H-imidazole-4- Yl) -ethyl] -carbamic acid benzyl ester;
(S)-[1-((4-Benzyloxy-benzyl)-{[(1-phenyl-cyclopentylmethyl) -carbamoyl] -methyl} -carbamoyl) -2- (1H-imidazole-4- Yl) -ethyl] -carbamic acid benzyl ester;
(S)-[1-((4-phenyl-benzyl)-{[(1-phenyl-cyclobutylmethyl) -carbamoyl] -methyl} -carbamoyl) -2- (1H-imidazol-4-yl ) -Ethyl] -carbamic acid benzyl ester;
(S)-[1-((4-methoxy-benzyl)-{[(1-phenyl-cyclobutylmethyl) -carbamoyl] -methyl} -carbamoyl) -2- (1H-imidazole-4- Yl) -ethyl] -carbamic acid benzyl ester;
(S)-[1-((4-Methyl-benzyl)-{[(1-phenyl-cyclobutylmethyl) -carbamoyl] -methyl} -carbamoyl) -2- (1H-imidazol-4-yl ) -Ethyl] -carbamic acid benzyl ester;
(S) -N- (4-benzyloxy-benzyl) -2- (3-benzyl-ureido) -3- (1H-imidazol-4-yl) -N-{[(1-phenyl-cyclobutyl Methyl) -carbamoyl] -methyl} -propionamide;
(S) -2- (3-benzyl-3-methyl-ureido) -N- (4-benzyloxy-benzyl) -3- (1H-imidazol-4-yl) -N-{[(1- Phenyl-cyclobutylmethyl) -carbamoyl] -methyl} -propionamide;
(S)-[1-((4-benzyloxy-benzyl)-{[(1-phenyl-cyclobutylmethyl) -carbamoyl] -methyl} -carbamoyl) -2- (1H-imidazole-4- Yl) -ethyl] -thiocarbamic acid S-benzyl ester;
(S)-(2- (1H-imidazol-4-yl) -1-{{[(1-phenyl-cyclobutylmethyl) -carbamoyl] -methyl}-[4- (pyridin-2-yl- Methoxy) -benzyl] -carbamoyl} -ethyl) -carbamic acid benzyl ester;
(S)-(1-((cyclohexyl-methyl)-{[(1-phenyl-cyclobutylmethyl) -carbamoyl] -methyl} -carbamoyl) -2- (1H-imidazol-4-yl) -Ethyl] -carbamic acid benzyl ester;
(S)-(1-((Isobutyl)-{[(1-phenyl-cyclobutylmethyl) -carbamoyl] -methyl} -carbamoyl) -2- (1H-imidazol-4-yl) -ethyl ] -Carbamic acid benzyl ester;
(S) -2- (3-benzyl-3-methyl-ureido) -3- (1H-imidazol-4-yl) -N- (4-methyl-benzyl) -N-{[(1-phenyl -Cyclobutylmethyl) -carbamoyl] -methyl} -propionamide;
(S)-[1-[(4-benzyloxy-benzyl)-({[1- (2, 6-dichlorophenyl) -cyclobutylmethyl] -carbamoyl} -methyl) -carbamoyl] -2- ( 1H-imidazol-4-yl) -ethyl] -carbamic acid benzyl ester;
[(S) -trans]-[1- (but-2-enyl-{[(1-phenyl-cyclobutylmethyl) -carbamoyl] -methyl} -carbamoyl) -2- (3H-imidazole-4 -Yl) -ethyl] -carbamic acid benzyl ester;
(S)-[2- (3H-imidazol-4-yl) -1-({[(1-phenyl-cyclobutylmethyl) -carbamoyl] -methyl} -propyl-carbamoyl) -ethyl] -carr Chest acid benzyl ester;
Benzyl N-((1S) -1- (1H-4-imidazolylmethyl) -2-oxo-2- (2-oxo-2-[(1-phenylcyclobutyl) methyl] aminoethyl) [(1R ) -1-phenylethyl] aminoethyl) carbamate;
(S)-[1-((1,1-Dimethyl-2-phenyl-ethyl)-{[(1-phenyl-cyclobutylmethyl) -carbamoyl] -methyl} -carbamoyl) -2- (3H- Imidazol-4-yl) -ethyl] -carbamic acid benzyl ester;
Benzyl N-((1S) -1- (1H-4-imidazolylmethyl) -2-oxo-2- (2-oxo-2-[(1-phenylcyclobutyl) methyl] aminoethyl) [(1S ) -1-phenylethyl] aminoethyl) carbamate;
Benzyl N-[(1S) -2-[(2-hydroxyethyl) (2-oxo-2-[(1-phenylcyclobutyl) methyl] aminoethyl) amino] -1- (1H-4-imida Zolylmethyl) -2-oxoethyl] carbamate;
3-[[(2S) -2-[(benzyloxy) carbonyl] amino-3- (1H-4-imidazolyl) propanoyl] (2-oxo-2-[(1-phenylcyclobutyl) Methyl] aminoethyl) amino] propanoic acid;
Methyl 3-[[(2S) -2-[(benzyloxy) carbonyl] amino-3- (1H-4-imidazolyl) propanoyl] (2-oxo-2-[(1-phenylcyclobutyl ) Methyl] aminoethyl) amino] propanoate;
Benzyl N-[(1S) -2-[(2-aminoethyl) (2-oxo-2-[(1-phenylcyclobutyl) methyl] aminoethyl) amino] -1- (1H-4-imidazolyl Methyl) -2-oxoethyl] carbamate;
Benzyl N- (1S) -1- (1H-4-imidazolylmethyl) -2-[[2- (methylamino) ethyl] (2-oxo-2-[(1-phenylcyclobutyl) -methyl] Aminoethyl) amino] -2-oxoethylcarbamate;
Benzyl N- (1S) -1- (1H-4-imidazolylmethyl) -2-[(2- (methoxyethyl) (2-oxo-2-[(1-phenylcyclobutyl) methyl] -amino Ethyl) amino] -2-oxoethylcarbamate;
Benzyl N- (1S) -1- (1H-4-imidazolylmethyl) -2-[(2- (morpholinoethyl) (2-oxo-2-[(1-phenylcyclobutyl) methyl]- Aminoethyl) amino] -2-oxoethylcarbamate;
Benzyl N- (1S) -1- (1H-4-imidazolylmethyl) -2-[(2- (methyl-2-phenylpropyl) (2-oxo-2-[(1-phenylcyclobutyl)- Methyl] aminoethyl) amino] -2-oxoethylcarbamate; and
1-phenylethyl N-[(1S) -2-[[4- (benzyloxy) -1,5-cyclohexadienyl] methyl (2-oxo-2-[(1-phenylcyclobutyl) -methyl] Aminoethyl) amino] -1- (1H-4-imidazolylmethyl) -2-oxoethyl] carbamate.
The present invention provides compounds of formula I and their pharmaceutically acceptable salts, esters, amides and 7 prodrugs thereof.
<Formula I>

In the above formula
R ^Q is

ego;
x is 0 or 1;
Each R ¹⁴ is independently hydrogen or C ₁ -C ₆ alkyl;
A is -COR ^a , -CO ₂ R ^{a '} , -CONHR ^a' , -CSR ^a , , , -C (S) OR ^{a '} , -C (S) NHR ^a' , -SO ₂ R ^a or -CONR ^a R ^{a ''} ;
R ^a , R ^{a '} and R ^a'' are independently C ₁ -C ₆ alkyl,-(CR ¹⁴ R ¹⁴ ) _m -cycloalkyl,-(CR ¹⁴ R ¹⁴ ) _m -aryl, or-(CR ¹⁴ R ¹⁴ ) _m -heteroaryl;
Each m is independently 0-3;
R ¹ , R ² and R ⁴ are independently hydrogen or C ₁ -C ₆ alkyl;
R³Is , C_One-C₆Alkyl, C₂-C₆Alkenyl,-(CR¹⁴R¹⁴)_m-Naphthyl,-(CH₂)_vCO₂R¹⁴,-(CH₂)_tNR¹⁴R¹⁴,-(CH₂)_v-O-C_One-C₆Alkyl,-(CH₂)_t-OH,-(CH₂)_tMorpholino,,,-(CR¹⁴R¹⁴)_m-(R^bPhenyl substituted by-or-(CR¹⁴R¹⁴)_m-(R^bHeteroaryl substituted with;
t is 2 to 6;
v is 1 to 6;
R ^b is —O-phenyl, —O-benzyl, halogen, C ₁ -C ₆ alkyl, hydrogen, —OC ₁ -C ₆ alkyl, -NH ₂ , -NHR ^a , -NR ^a R ^{a '} , , , , -OH, , , -O (CH ₂ ) _y NR ^a R ^{a '} , -CF ₃ , -NO ₂ , , , -CN, -OPO ₃ H ₂ , -CH ₂ PO ₃ H ₂ , , -N ₃ , -CF ₂ CF ₃ , -SO ₂ R ^a , -SO ₂ NR ^a R ^{a '} , -CHO, -O (CH ₂ ) _m -aryl, -O (CH ₂ ) _m -cycloalkyl, -(CH ₂ ) _m -aryl,-(CH ₂ ) _m -cycloalkyl,-(CH ₂ ) _m -heteroaryl, -CH = CHC ₆ H ₅ , -OCOCH ₃ , or -O (CH ₂ ) _m- Heteroaryl;
y is 2 or 3;
R ⁵ is

ego;
Each n is independently 2, 3 or 4;
R ⁱ , R ^g and R ^h are independently hydrogen, halogen, -OC ₁ -C ₆ alkyl, C ₁ -C ₆ alkyl, -CN, -OPO ₃ H ₂ , -CH ₂ PO ₃ H ₂ , -O- Phenyl, -O-benzyl, , , , , -O (CH ₂ ) _y NR ^a R ^{a '} , -NH ₂ , -NHR ^a , -NR ^a R ^a' , , , -OH, CF ₃ , -NO ₂ , , , , -N ₃ , -CF ₂ CF ₃ , -SO ₂ R ^a , -SO ₂ NR ^a R ^{a '} , -CHO or -OCOCH ₃ ;
R ^c and R ^d are independently C ₁ -C ₆ alkyl, — (CH ₂ ) _m -cycloalkyl or hydrogen.
The present invention also provides compounds of formula (II) and their pharmaceutically acceptable salts, esters, amides and prodrugs thereof
<Formula II>

In the above formula
R ⁶ is -O-benzyl, , -NH-benzyl, -N (C ₁ -C ₆ alkyl) -benzyl or -SCH ₂ -phenyl;
R ⁸ is hydrogen, halogen, C ₁ -C ₆ alkyl, —O-benzyl, —OCH ₂ -pyridyl, —OC ₁ -C ₆ alkyl, —CF ₃ , —OH or —phenyl;
R ¹² is ego;
R ¹⁰ and R ¹³ are independently hydrogen or C ₁ -C ₆ alkyl;
n is 2, 3 or 4;
R ¹⁴ is hydrogen or methyl;
R^j, R^kAnd R^lIndependently hydrogen, halogen, -NH₂, -NHR^a, -OC_One-C₆Alkyl or -C_One-C₆Alkyl.
The invention also provides compounds of formula III and their pharmaceutically acceptable salts, esters, amides and prodrugs thereof
<Formula III>

In the above formula
Each n is 2, 3 or 4;
X is NH, O, or -NCH ₃ ;
R ¹⁵ is —O-benzyl, —CF ₃ , hydrogen, halogen, —OH, —phenyl, —C ₁ -C ₆ alkyl, —OCH ₂ -pyridyl or —OC ₁ -C ₆ alkyl.
The present invention also provides a compound of formula IV
<Formula IV>

In the above formula
X is NH, O, or -NCH ₃ ;
R ^m is C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl,-(CH ₂ ) _t NR ¹⁴ R ¹⁴ ,-(CH ₂ ) _v -OC ₁ -C ₆ alkyl,-(CH ₂ ) _t- OH,-(CH ₂ ) _t -morpholino, , ,-(CH ₂ ) _v -CO ₂ R ¹⁴ or ego;
Each R ¹⁴ is independently hydrogen or —C ₁ -C ₆ alkyl;
n is 2, 3 or 4;
m is 0 to 3;
t is 2 to 6;
v is 1-6.
The term "alkyl" means straight or branched chain hydrocarbons having 1 to 6 carbon atoms, for example methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n Pentyl, n-hexyl and the like.
The term "cycloalkyl" refers to a saturated hydrocarbon ring having 3 to 7 carbon atoms and includes, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, adamantyl and the like.
The term "aryl" refers to alkyl, O-alkyl and S-alkyl, 0H, SH, F, Cl, Br, I, CF ₃ , NO ₂ , NH ₂ , NHCH ₃ , N (CH ₃ ) ₂ , NHCO-alkyl , -OCH ₂ C ₆ H ₅ , (CH ₂ ) _m CO ₂ H, (CH ₂ ) _m CO ₂ -alkyl, (CH ₂ ) _m SO ₃ H, (CH ₂ ) _m PO ₃ H ₂ , (CH ₂ ) phenyl, 5-fluore, unsubstituted or substituted with 1 to 3 substituents selected from _m PO ₃ (alkyl) ₂ , (CH ₂ ) _m SO ₂ NH ₂ and (CH ₂ ) _m SO ₂ NH-alkyl An aromatic ring that is a nil, 1-naphthyl, or 2-naphthyl group, wherein alkyl is defined as above and m is 0, 1, 2 or 3.
The term "heteroaryl" refers to 2- or 3-thienyl, 2- or 3-furanyl, 2- or 3-, unsubstituted or substituted with one or two substituents of the substituent group mentioned for aryl above. Heteroaromatic ring that is a pyrrolyl, 2-, 3- or 4-pyridyl, imidazolyl, 2-, 3-, 4-, 5-, 6- or 7-indolyl group.
The symbol "-" means a bond.
The term "patient" means all animals, including humans. Examples of patients include humans, cows, dogs, cats, goats, sheep and pigs.
By "therapeutically effective amount" is meant an amount of a compound of the invention that, when administered to a patient, ameliorates the symptoms of or prevents restenosis. A therapeutically effective amount of a compound of the present invention can be readily determined by one skilled in the art by observing the result after administering an amount of the compound to a patient. Those skilled in the art are also familiar with identifying patients with cancer or restenosis or those at risk of developing restenosis.
The term "cancer"
breast;
ovary;
cervix;
prostate;
Testes;
esophagus;
Glioblastoma;
Neuroblastoma;
top;
Cutaneous keratinocytes;
Lung, epidermoid cancer, large cell cancer, adenocarcinoma;
goal;
Colon, adenocarcinoma, adenoma;
Pancreas, adenocarcinoma;
Thyroid, vesicle cancer, undifferentiated cancer, papillary cancer;
Normal somatoma;
Melanoma;
sarcoma;
Bladder cancer;
Liver cancer and gallbladder pathways;
Kidney cancer;
Myeloid disease;
Lymphoid disease, hochkin, hair cells;
Narrowing and orbital (oral), labia, tongue, oral cavity, pharynx;
Intestine;
Colon-rectal, large intestine, rectum;
Brain and central nervous system; And
leukemia
Cancer, but is not limited to.
The term “pharmaceutically acceptable salts, esters, amides and prodrugs”, as used herein, refers to amphoteric forms of the compounds of the present invention, as well as possible, as well as carboxylate salts, amino acids of the compounds of the present invention. Addition salts, esters, amides and prodrugs, suitable for use in contact with patient tissues without involving undue toxicity, irritation, allergic reactions, etc., within the scope of normal pharmaceutical judgment, and with a reasonable benefit / risk ratio, Means to be effective as intended. The term "salt" means an inorganic or organic acid addition salt of a compound of the invention that is relatively nontoxic. Such salts can be prepared on-the-fly in the final isolation and purification of the compound or by isolating the salts formed after the purified compound in free base form is reacted separately with a suitable organic or inorganic acid. Representative salts include bromic acid, hydrochloric acid, sulfate, bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, Citrate, maleate, fumarate, succinate, tartrate, naphtholate mesylate, glucoheptonate, lactobionate and laurylsulfonate salts and the like. These include, but are not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, etc. Non-toxic ammonium, quaternary ammonium and amine cations In addition to these, it includes cations based on alkali or alkaline earth metals such as sodium, lithium, potassium, calcium, magnesium and the like (SM Berge et al., Which is incorporated herein by reference for example). Salts ", J. Pharm. Sci., 1977; 66: 1-19.)
Examples of pharmaceutically acceptable non-toxic esters of the compounds of the invention include C ₁ -C ₆ alkyl esters wherein the alkyl group is straight or branched. Acceptable esters also include C ₅ -C ₇ cycloalkyl esters in addition to arylalkyl esters such as, but not limited to, benzyl. Preference is given to C ₁ -C ₄ alkyl esters. Esters of the compounds of the present invention can be prepared according to conventional methods.
Examples of pharmaceutically acceptable non-toxic amides of the compounds of the present invention include ammonia, amides derived from primary C ₁ -C ₆ alkyl amines with linear or branched alkyl groups and secondary C ₁ -C ₆ dialkyl amines. do. In the case of secondary amines, the amine may be in the form of a 5- or 6-membered heterocycle containing one nitrogen atom. Preferred are amides derived from ammonia, C ₁ -C ₃ alkyl primary amines and C ₁ -C ₂ dialkyl secondary amines. Amides of the compounds of the present invention can be prepared according to conventional methods.
The term “prodrug” refers to a compound that converts rapidly in vivo and, for example, by hydrolysis in blood, to produce the parent compound of the agent. tea. T. Higuchi and V. V. Stella, A. See you. s. "Pro-drugs as Novel Delivery Systems" in Volume 14 of the A.C.S. Symposium Series and Biereversible Carriers in Drug Design, ed. Edward B. Edward B. Roche, American Pharmaceutical Association and Pergamon Publishing, 1987, which is incorporated by reference in their entirety.
The compounds of the present invention can be administered to a patient alone or as part of a composition containing other ingredients such as excipients, diluents and carriers known in the art. The composition can be orally, rectally, parenterally (intravenously, intramuscularly or subcutaneously), intramurally, intravaginally, intraperitoneally, in the bladder, locally (powder, ointment or drop), or buccal or cost It can be administered to humans and animals as a nebulizer.
Compositions suitable for parenteral injection include physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile infusion solutions or dispersions. Examples of suitable aqueous or non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, etc.), suitable mixtures thereof, vegetable oils (such as olive oil) and ethyl oleate Injectable organic esters. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the desired particle size in the case of dispersions, and by the use of surfactants.
Such compositions may also include adjuvant such as preservatives, wetting agents, emulsifiers and administration agents. The action of microorganisms can be inhibited by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid and the like. It may also be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like. Absorption of the injectable pharmaceutical form may be prolonged by the use of absorption retardants such as, for example, aluminum monostearate and gelatin.
Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is a conventional inert excipient (or carrier) such as sodium citrate or dicalcium phosphate, or (a) for example starch, lactose, sucrose, glucose, mannitol and silicic acid Fillers or extenders; (b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) supplements such as, for example, glycerol; (d) disintegrants such as, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicate complexes and sodium carbonate; (e) solution coagulants such as, for example, paraffin; (f) absorption accelerators such as, for example, quaternary ammonium compounds; (g) wetting agents, for example cetyl alcohol and glycerol monostearate; (h) adsorbents such as, for example, kaolin and bentonite; And (i) lubricants such as, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate or mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise a buffer.
Solid compositions of a similar type may also be used as fillers in soft or hard-filled gelatin capsules using excipients such as lactose or lactose in addition to high molecular weight polyethylene glycols and the like.
Solid dosage formulations such as tablets, dragees, capsules, pills, and granules can be prepared with enteric coatings and other coatings and shells known in the art. They may contain opacifying agents and may also be compositions which release the active compound or compounds in a delayed manner in certain parts of the intestinal tract. Examples of embedding compositions that can be used are polymeric substances and waxes. The active compound may be in the form of microcapsules and, where appropriate, with one or more of the above excipients.
Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. Liquid dosage forms may contain, in addition to the active compound, inert excipients such as water or other solvents, solubilizers and emulsifiers commonly used in the art, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl Alcohols, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol, tetrahydrofurfuryl alcohol, cremofo EL (derivatives of castor oil and ethylene oxide; purchased from Sigma Chemical Co., St. Louis, MO), fatty acid esters of polyethylene glycol and sorbitan, or mixtures of these materials.
In addition to these inert diluents, the compositions may also include auxiliaries such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents and fragrances.
Suspensions may be used in addition to the active materials, for example ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxy, bentonite, aga-agar and tragacanth, or Suspending agents, such as a mixture, can be contained.
Rectal compositions are preferably suppositories, or suppository waxes, which can be prepared by mixing the compounds of the present invention with suitable non-irritating excipients or carriers, such as cocoa butter and polyethylene glycol, which are solid or body temperature at normal temperatures. Esau is a liquid that dissolves in the rectum or vagina to release the active compound.
Formulations for topical administration of a compound of this invention include ointments, powders, sprays and inhalants. The active compound is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required. Ophthalmic formulations, eye ointments, powders and solutions are also intended to fall within the scope of the present invention.
Compounds of the invention may be administered to a patient at a dosage level in the range of about 0.1 to about 2,000 mg per day. For normal adult humans having a weight of about 70 kg, administration in the range of about 0.01 to about 100 mg per kg of body weight per day is preferred. However, specific dosages may vary. For example, the dosage will depend on several factors including the patient's requirement, the severity of the condition being treated and the pharmacological activity of the compound used. Determination of the optimal dose for a particular patient is known to those skilled in the art.
The compounds of the present invention may exist in different stereoisomeric forms due to the asymmetric centers present in the compounds. All stereoisomeric forms, including mixtures of stereoisomers as well as racemic mixtures of the present compounds, are intended to constitute part of the present invention.
In addition, the compounds of the present invention may exist in unsolvated forms in addition to solvated forms by pharmaceutically acceptable solvents such as water and ethanol. In general, the solvated forms are considered equivalent to the unsolvated forms for the purposes of the present invention.
The following examples are intended to illustrate certain embodiments of the invention and are not intended to limit the scope of the specification or claims in any way.
Scheme 1 shows a general method for preparing the compounds of the present invention.
Scheme 1
Preparation of Example 1

Scheme 2
Preparation of Example 10

In step 4 of Scheme 1, a surrogate polymer PyBOP; Dicyclohexylcarbodiimide (DCC) / 1-hydroxy-benzotriazole (HOBt); O-benzotriazol-1-yl-N, N, N ', N'-tetra-methyluronium hexafluorophosphate (HBTU); And O- (7-azabenzotriazol-1yl) -N, N, N ', N'-tetramethyluronium hexafluorophosphate (HATU) / 1-hydroxy-7-azabenzotriazole (HOAt ) Can also be used. In step 5, 90 ° C., 80% acetic acid / water may be used for 30 minutes.
Abbreviation
DCM dichloromethane
Na (OAc) ₃ BH Sodium Triacetoxyborohydride
PyBOP Benzotriazol-1-yl-oxy-tris-pyrrolidino-phosphonium
Hexafluorophosphate
DIEA diisopropylethylamine
THF tetrahydrofuran
BOP Benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium
Hexafluorophosphate
TFA trifluoroacetic acid
EA ethyl acetate
<Example 1>
(S)-[1-((4-benzyloxy-benzyl)-{[1- (phenyl-cyclobutylmethyl) -carbamoyl] -methyl} -carbamoyl) -2- (1H-imidazole-4- Yl) -ethyl] -carbamic acid benzyl ester
Step 1: [(4-benzyloxy-benzyl) -amino] -acetic acid methyl ester
To a suspension of glycine methyl ester hydrochloric acid (1.26 g, 10 mmol) and 4-benzyloxybenzaldehyde (2.12 g, 10 mmol) in DCM (50 mL) was added Na (OAc) ₃ BH (3.81 g, 15 mmol) at 0 ° C. under nitrogen. The suspension was allowed to warm to room temperature and stirred for 4 hours. The suspension was poured into saturated aqueous NaHCO ₃ solution and the layers separated. The aqueous layer was extracted with DCM (4 × 25 mL). The combined organic extracts were dried over MgSO ₄ and concentrated. Flash chromatography (EA) gave 1.15 g (40.4%) of the title compound as a white solid; Melting point 57-58 ° C.
Analytical calculation of C ₁₇ H ₁₉ NO ₃ :
C, 71.56; H, 6.71; N, 4.91.
Found C, 71.45; H, 6. 99; N, 4.92.
Mass Spectrum (MS)-Chemical Ionization (CI):
Calcd for M + 1: 286; Found: 286.
NMR spectra matched the structure.
Step 2: (S)-{(4-benzyloxy-benzyl)-[2-benzyloxycarbonylamino-3- (1-trityl-1 H-imidazol-4-yl) -propionyl] -amino} Acetic acid methyl ester
(S) -2-benzyloxycarbonylamino-3- (1-trityl-1H-imidazol-4-yl) -propionic acid [Cbz-His (Trt)] (Hedspes J. in DCM (100 mL). Hudspeth JP, Kaltenbronn JS, Repine JT, Roark WH, Stier MA [Renin Inhibitors III] U.S. Patent No. 4,735,933; 1988) (5.85 g, 11 mmol) and PyBOP (5.72 g, 11 mmol) were added to the methyl ester of step 1 (2.85 g, 10 mmol) at 0 ° C. followed by DIEA ( 3.05 mL, 17.5 mmol) was added. The solution was warmed to room temperature and stirred for 5 hours under nitrogen. The solution was poured into saturated aqueous NaHCO ₃ solution and the layers separated. The aqueous layer was extracted with DCM (4 × 50 mL). The combined organic layer was dried over MgSO ₄ and concentrated. Flash chromatography (10% methanol [MeOH] in DCM) gave 4.48 g (55.4%) of the title compound as a white foam.
C ₅₀ H ₄₆ N ₄ O ₆ Analytical calculations for 0.5 H ₂ O:
C, 74.33; H, 5. 86; N, 6.93.
Found: C, 74.00; H, 5.75; N, 6.79.
MS-electrospray (ES): calcd for M + 1: 799.3; Found: 799.3.
NMR spectra matched the structure.
Step 3: (S)-{(4-benzyloxy-benzyl)-[2-benzyloxycarbonylamino-3- (1-trityl-1 H-imidazol-4-yl) -propionyl] -amino} Acetic acid
To the solution of methyl ester (0.799 g, 1 mmol) of step 2 in THF (10 mL) was added H ₂ O (3.3 mL) to form a solution. The solution was cooled to 0 ° C. and treated with LiOH.H ₂ O (0.050 g, 1.2 mmol). The solution was warmed to room temperature and stirred for 4 hours. Further LiOH.H ₂ O (0.050 g, 1.2 mmol) was added and the suspension was further stirred for 1.5 hours. The suspension was concentrated and diluted with H ₂ O and adjusted to pH = 2 (paper) with 1M HCl. The suspension was extracted with EA (4x20 mL). The organic extract was dried over MgSO ₄ and concentrated. Flash chromatography (10% MeOH in DCM) gave 0.706 g (89.0%) of the title compound as a white foam.
C ₄₉ H ₄₄ N ₄ O ₆ Analytical calculations for 0.5 H ₂ O:
C, 74.13; H, 5.71; N, 7.05.
Found: C, 74.22; H, 5. 70; N, 6.81.
MS-ES: calcd for M + 1: 785.3; Found: 785.2.
NMR and IR spectra matched the structure.
Step 4: (S)-[1-((4-Benzyloxy-benzyl)-{[(1-phenyl-cyclobutylmethyl) -carbamoyl] -methyl} -carbamoyl) -2- (1-trityl) -1H-imidazol-4-yl) -ethyl] carbamic acid benzyl ester
Acid (0.500 g, 0.637 mmol), BOP (0.310 g, 0.70 mmol) and C- (1-phenyl-cyclobutyl) -methylamine hydrochloric acid (Bridge AJ) in DCM (10 mL) Hamilton H., Moos WH, and Szotek DL, "N ⁶ -Substituted Adenosines," US Patent No. 4,755,594; 1988) 0.139 g, 0.701 mmol) was added DIEA (0.29 mL, 1.75 mmol) at 0 ° C. The resulting solution was stirred at rt overnight. The solution was poured into saturated aqueous NaHCO ₃ solution and the layers separated. The aqueous layer was extracted with DCM (3 × 50 mL). The mixed organic layer was washed with H ₂ O, then washed twice with 0.5 M HCl, once again with saturated aqueous NaHCO ₃ solution, dried over MgSO ₄ , and concentrated. Flash chromatography (10% MeOH in DCM) gave 0.49 g of the title compound as a white foam which was used directly in the next reaction without characterization.
Step 5: (S)-[1-((4-Benzyloxy-benzyl)-{[(1-phenyl-cyclobutylmethyl) -carbamoyl] -methyl} -carbamoyl) -2- (1H-imidazole -4-yl) -ethyl] -carbamic acid benzyl ester
To a solution of the trityl compound (0.49 g) of step 4 in DCM (10 mL) was added TFA (10 mL). The solution was stirred at rt for 2 h and then concentrated. The residue was taken up in DCM and washed with saturated aqueous NaHCO ₃ . The aqueous layer was extracted with DCM (2 × 50 mL). The combined organic extracts were dried over MgSO ₄ and concentrated. Flash chromatography (10% MeOH in DCM) gave 0.22 g (50% yield in 2 steps) of the title compound as a white foam.
C ₄₁ H ₄₃ N ₅ O ₅ Analytical calculations for 0.5H ₂ O:
C, 70.87; H, 6. 38; N, 10.08.
Found: C, 70.81; H, 6. 46; N, 9.91.
MS-atmospheric pressure chemical ionization (APCI):
Calcd for M + 1: 686.3; Found: 686.3.
NMR and IR spectra matched the structure.
<Example 2>
(S)-[1-((4-benzyloxy-benzyl)-{[(1-phenyl-cyclopropylmethyl) -carbamoyl] -methyl} -carbamoyl) -2- (1H-imidazole-4- Yl) -ethyl] -carbamic acid benzyl ester
Example 1, steps 4 and 5, replaced by C- (1-phenyl-cyclopropyl) -methylamine hydrochloric acid (US Pat. No. 4,755,594; 1988) instead of C- (1-phenyl-cyclobutyl) -methylamine hydrochloric acid This gave 0.11 g (26% yield over 2 steps) of the title compound as a white foam.
C ₄₀ H ₄₁ N ₅ O ₅ Analytical calculations for 0.5H ₂ O:
C, 70.57; H, 6. 22; N, 10.29.
Found: C, 70.43; H, 5.98; N, 10.25.
MS-APCI: Calcd for M + 1: 672.3; Found: 672.3.
NMR and IR spectra matched the structure.
<Example 3>
(S)-(2- (1H-imidazol-4-yl) -1-{{[(1-phenyl-cyclobutylmethyl) -carbamoyl] -methyl}-[4- (pyridin-2-ylmethoxy ) -Benzyl] -carbamoyl} -ethyl-carbamic acid benzyl ester
Step 1: [(4- (pyridin-2-ylmethoxy) -benzyl) -amino] -acetic acid methyl ester
In Example 1, Step 1, the title compound was replaced by oil by replacing 4-benzyloxybenzaldehyde with 4- (2-pyridinylmethoxy) benzaldehyde (J. Het. Chem., 1988; 25: 129). Obtained (36.3% yield).
MS-CI: calcd for M + 1: 287; Found: 287.
Step 2: (S)-{(4- (pyridin-2-ylmethoxy) -benzyl)-[2-benzyloxycarbonylamino-3- (1-trityl-1H-imidazol-4-yl)- Propionyl] -amino} -acetic acid methyl ester
In Example 1, step 2, the title compound was obtained as a white foam by replacing the compound of step 1 above with [(4-benzyloxy-benzyl) -amino] -acetic acid methyl ester (90.1% yield).
MS-APCI: Calcd for M + 1: 800.3; Found: 800.3.
Step 3: (S)-{(4- (pyridin-2-ylmethoxy) -benzyl)-[2-benzyloxycarbonylamino-3- (1-trityl-1H-imidazol-4-yl)- Propionyl] -amino} -acetic acid
In Example 1, step 3, (S)-{(4-benzyloxy-benzyl)-[2-benzyloxycarbonylamino-3- (1-trityl-1H-imidazol-4-yl) -propy The title compound was obtained as a white foam (87.5% yield) by replacing with onyl] -amino} -acetic acid methyl ester with the methyl ester of step 2 above.
MS-APCI: Calcd for M + 1: 786.3; Found: 786.3.
Step 4: (S)-[1-(((4-Pyridin-2-ylmethoxy) -benzyl)-{[(1-phenyl-cyclobutylmethyl) -carbamoyl] -methyl} -carbamoyl) -2 -(1-trityl-1H-imidazol-4-yl) -ethyl] -carbamic acid benzyl ester
In Example 1, step 4, (S)-{(4-benzyloxy-benzyl)-[2-benzyloxycarbonylamino-3- (1-trityl-1H-imidazol-4-yl) -propy The title compound was obtained as a clear oil by replacing the acid of step 3 above with onyl] -amino} -acetic acid (100% yield).
MS-APCI: Calcd for M + 1: 929.4; Found: 929.2.
Step 5: (S)-(2- (1H-imidazol-4-yl) -1-{{[1-phenyl-cyclobutylmethyl) -carbamoyl] -methyl}-[4- (pyridine-2- Monomethoxy) -benzyl] -carbamoyl} -ethyl) -carbamic acid benzyl ester
In Example 1, step 5, (S)-[1-((4-benzyloxy-benzyl)-{[(1-phenyl-cyclobutylmethyl) -carbamoyl] -methyl} -carbamoyl) -2- The title compound was obtained as a white foam by replacing the compound of step 4 above with (1-trityl-1H-imidazol-4-yl) -ethyl] -carbamic acid benzyl ester (87% yield).
C ₄₀ H ₄₂ N ₆ O ₅ ㆍ 0.25 Analytical calculated value of DCM:
C, 68.28; H, 6.05; N, 11.87.
Found: C, 68.23; H, 6.05; N, 11.90.
MS-APCI: Calcd for M + 1: 687.3; Found: 687.2.
<Example 4>
(S)-[1-((4-Methyl-benzyl)-{[(1-phenyl-cyclobutylmethyl) -carbamoyl] -methyl} -carbamoyl) -2- (1H-imidazol-4-yl ) -Ethyl] -carbamic acid benzyl ester
Step 1: [(4-Methyl-benzyl) -amino] -acetic acid methyl ester
In Example 1, step 1, the title compound was obtained as a colorless oil by replacing 4-methylbenzaldehyde instead of 4-benzyloxybenzaldehyde (53% yield).
MS-CI: calcd for M + 1: 194; Found: 194.
Step 2: (S)-{(4-Methyl-benzyl)-[2-benzyloxycarbonylamino-3- (1-trityl-1 H-imidazol-4-yl) -propionyl] -amino}- Acetic acid methyl ester
In Example 1, step 2, the title compound was obtained as a white foam by replacing the compound of step 1 above with [(4-benzyloxy-benzyl) -amino] -acetic acid methyl ester (71% yield).
MS-APCI: Calcd for M + 1: 707.3; Found: 706.3.
Step 3: (S)-{(4-Methyl-benzyl)-[2-benzyloxycarbonylamino-3- (1-trityl-1 H-imidazol-4-yl) -propionyl] -amino}- Acetic acid
In Example 1, step 3, (S)-{(4-benzyloxy-benzyl)-[2-benzyloxycarbonylamino-3- (1-trityl-1H-imidazol-4-yl) -propy The title compound was obtained as a white foam by replacing the compound of step 2 above with onyl] -amino} -acetic acid methyl ester (100% yield).
MS-APCI: Calcd for M + 1: 693.3; Found: 693.2.
Step 4: (S)-[1-((4-Methyl-benzyl)-{[(1-phenyl-cyclobutylmethyl) -carbamoyl] -methyl} -carbamoyl) -2- (1-trityl- 1H-imidazol-4-yl) -ethyl] -carbamic acid benzyl ester
In Example 1, step 4, (S)-{(4-benzyloxy-benzyl)-[2-benzyloxycarbonylamino-3- (1-trityl-1H-imidazol-4-yl) -propy The title compound was obtained as a white foam by replacing the compound of step 3 above with onyl] -amino} -acetic acid (67% yield).
MS-APCI: Calcd for M + 1: 836.4; Found: 836.1.
Step 5: (S)-[1-((4-Methyl-benzyl)-{[(1-phenyl-cyclobutylmethyl) -carbamoyl] -methyl} -carbamoyl) -2- (1H-imidazole- 4-yl) -ethyl] -carbamic acid benzyl ester
In Example 1, step 5, (S)-[1-((4-benzyloxy-benzyl)-{[(1-phenyl-cyclobutylmethyl) -carbamoyl] -methyl} -carbamoyl) -2- The title compound was obtained as a white foam (55% yield) by replacing the compound of step 4 above with (1-trityl-1H-imidazol-4-yl) -ethyl] -carbamic acid benzyl ester.
C ₃₅ H ₃₉ N ₅ O ₄ Analytical calculations for 0.13 DCM:
C, 69.77; H, 6. 54; N, 11.58.
Found: C, 69.77; H, 6. 38; N, 11.52.
MS-APCI: Calcd for M + 1: 594.3; Found: 594.2.
Example 5
(S)-[1-((4-Benzyloxy-benzyl)-{[(1-phenyl-cyclopentylmethyl) -carbamoyl] -methyl} -carbamoyl) -2- (1H-imidazole-4- Yl) -ethyl] -carbamic acid benzyl ester
In Examples 1, 4 and 5, replaced by C- (1-phenyl-cyclopentyl) -methylamine hydrochloric acid (US Pat. No. 4,755,594; 1988) instead of C- (1-phenyl-cyclobutyl) -methylamine hydrochloric acid This gave 0.28 g of the title compound as a white foam (62% yield over two steps).
C ₄₂ H ₄₅ N ₅ O ₅ Analytical calculations for 0.5 H ₂ O:
C, 71.17; H, 6. 54; N, 9.88.
Found: C, 71.44; H, 6. 43; N, 9.93.
MS-APCI: Calcd for M + 1: 700.3; Found: 700.3.
<Example 6>
(S)-[1-((4-methoxy-benzyl)-{[(1-phenyl-cyclobutylmethyl) -carbamoyl] -methyl} -carbamoyl) -2- (1H-imidazole-4- Il) -ethyl] -carbamic acid benzyl ester was synthesized with the change of removing trityl protecting groups before forming the C-terminal amide in Scheme 1.
Step 1: [(4-methoxy-benzyl) -amino] -acetic acid tert-butyl ester
In Example 1, step 1, the title compound was obtained as a yellow oil by replacing 4-methoxybenzaldehyde instead of 4-benzyloxybenzaldehyde and glycine tert-butyl ester hydrochloric acid instead of glycine methyl ester hydrochloric acid (80% yield).
Step 2: (S)-{(4-methoxy-benzyl)-[2-benzyloxycarbonylamino-3- (1-trityl-1 H-imidazol-4-yl) -propionyl] -amino} Acetic acid tert-butyl ester
In Example 1, step 2, the title compound was obtained as a white foam by replacing the compound of step 1 above with [(4-benzyloxy-benzyl) -amino] -acetic acid methyl ester (73% yield).
Step 3: (S)-{(4-methoxy-benzyl)-[2-benzyloxycarbonylamino-3- (1H-imidazol-4-yl) -propionyl] -amino} -acetic acid
To the solution of the trityl compound of step 2 (3.93 g, 5.14 mmol) in DCM (25 mL) was added TFA (25 mL). The solution was stirred at rt for 3 h and then concentrated. The residue was added to ether (300 mL) and cooled. Filtration gave the white taryl solid to afford 2.37 g (99% yield) of the title compound.
Step 4: (S)-[1-((4-methoxy-benzyl)-{[(1-phenyl-cyclobutylmethyl) -carbamoyl] -methyl} -carbamoyl) -2- (1H-imidazole -4-yl) -ethyl] -carbamic acid benzyl ester
In Example 1, step 4, (S)-{(4-benzyloxy-benzyl)-[2-benzyloxycarbonylamino-3- (1-trityl-1H-imidazol-4-yl) -propy The title compound was obtained as a white foam by replacing the compound of step 3 above with onyl] -amino} -acetic acid (17% yield). Final purification was done by preparative C18 reverse phase high pressure liquid chromatography using 0.1% TFA / acetonitrile gradient in 0.1% TFA / H ₂ O.
C ₃₅ H ₃₉ N ₅ O ₅ Analytical calculations for 1.13 TFA and 0.4 H ₂ O:
C, 59.99; H, 5.53; N, 9.39.
Found: C, 59.99; H, 5.53; N, 9.26.
MS-APCI: Calcd for M + 1: 610.3; Found: 610.3.
<Example 7>
(S) -2- (3-benzyl-3-methyl-ureido) -3- (1H-imidazol-4-yl) -N- (4-methyl-benzyl) -N-{[(1-phenyl -Cyclobutylmethyl) -carbamoyl] -methyl} -propionamide
The following changes were made to Example 1 to prepare the title compound: In step 1, 4-methylbenzaldehyde was substituted for 4-benzyloxybenzaldehyde; In step 2, replace with N-methyl-N-benzyl-urea-histidine (trityl) (steps 1 and 2 below) instead of Cbz-His- (Trt); In step 3, 1N NaOH in methanol / THF is used instead of LiOH: H ₂ 0 in THF; In step 4, using DCC / HOBt instead of BOP as binder; In step 5, 80% acetic acid aqueous solution was used instead of 50% TFA in DCM. The title compound was obtained as white foam; 0.16 g (57% yield).
C ₃₆ H ₄₂ N ₆ O ₃ : Analytical calculations for 0.16 CH ₂ Cl ₂ :
C, 70.01; H, 6.88; N, 13.55.
Found: C, 70.04; H, 6. 86; N, 13.62.
MS-APCI: Calcd for M + 1: 607.8; Found: 607.2.
Step 1: N-methyl-N-benzyl-urea-histidine- (trityl) methyl ester
Histidine- (trityl) methyl ester hydrochloric acid (2.0 g, 4.2 mmol) was suspended in DCM (20 mL) and the solution washed twice with saturated NaHCO ₃ , brine, dried over MgSO ₄ and cooled to 0 ° C. Triethylamine (0.65 mL, 8.8 mmol) and 4-nitrophenyl chloroformate (0.93 g, 4.7 mmol) were added. The reaction was stirred at 0 ° C. under nitrogen for 1.5 h. Then N-benzyl-N-methylamine (1.14 mL, 8.8 mmol) in DCM (10 mL) was added slowly and the reaction was stirred overnight at room temperature under nitrogen. After removing the solvent ethyl acetate was added to the residue. The organic solution was washed twice with water, saturated NaHCO ₃ , brine, dried over MgSO ₄ and concentrated. Foam was obtained by chromatography using 1: 1 ethyl acetate: hexanes; 1.19 g (50% yield).
Step 2: N-methyl-N-benzyl-urea-histidine- (trityl)
The methyl ester of step 1 (1.19 g, 2.1 mmol) was dissolved in THF: methanol (10 mL each). NaOH (1N) (6.3 mL, 6.3 mmol) was added and the reaction was stirred overnight. Solvent was removed. HCl (IN) (6.3 mL) was added and the product extracted with ethyl acetate. The organic solution was then washed twice with brine, dried over MgSO ₄ and concentrated to give a white foam; 1.4 g (100% yield).
<Example 8>
(S) -2- (3-benzyl-3-methyl-ureido) -N- (4-benzyloxy-benzyl) -3- (1H-imidazol-4-yl) -N-{[(1- Phenyl-cyclobutylmethyl) -carbamoyl] -methyl} -propionamide
The title compound was prepared by adding the following changes to Example 1: In step 2, instead of Cbz-His- (Trt), N-methyl-N-benzyl-urea-histidine (trityl) (Example 7, step 1 And 2); In step 3, 1N NaOH in methanol / THF is used instead of LiOH: H ₂ 0 in THF; In step 4, using DCC / HOBt instead of BOP as binder; In step 5, 80% acetic acid aqueous solution was used instead of 50% TFA in DCM. The title compound was obtained as white foam; 0.12 g (52% yield).
C ₄₂ H ₄₆ N ₆ O ₄ : Analytical calculation for 0.33 CH ₂ Cl ₂ :
C, 68.87; H, 6. 33; N, 11.39.
Found: C, 68.87; H, 6. 43; N, 11.11.
MS-APCI: Calcd for M + 1: 699.9; Found: 699.4.
Example 9
(S)-[1-[(4-benzyloxy-benzyl)-({[1- (2,6-dichlorophenyl) -cyclobutylmethyl] -carbamoyl} -methyl) -carbamoyl] -2- ( 1H-imidazol-4-yl) -ethyl] -carbamic acid benzyl ester
The following changes were made to Example 1 to prepare the title compound: In step 3, 1N NaOH in methanol / THF was used instead of LiOH: H ₂ O in THF; In step 4, [1- (2,6-dichlorophenyl) cyclobutyl] methylamine hydrochloric acid (step 1 below) was replaced by C- (1-phenyl-cyclobutyl) methylamine hydrochloric acid. The title compound was purified by reverse phase high pressure liquid chromatography (eluant: 0.1% TFA aqueous solution and 0.1% TFA in acetonitrile) to give a white foam; 0.015 g (5% yield).
Analytical calculation of C ₄₁ H ₃₉ N ₅ O ₅ Cl ₂ : 1.68 C ₂ H ₁ O ₂ F ₃ : 1.01 H ₂ O:
C, 55.36; H, 4. 47; N, 7.28.
Found: C, 55.36; H, 4. 47; N, 7.26.
MS-APCI: Calcd for M + 1: 752.2; Found: 752.6.
Step 1: [1- (2,6-dichlorophenyl) cyclobutyl] methylamine hydrochloric acid
1- (2,6-dichlorophenyl) -1-cyclobutanecarbonitrile (1 g, 4.4 mmol) was reduced to Raney nickel in methanol / NH ₃ . The catalyst was removed and washed with methanol. The filtrate was concentrated and diethyl ether (100 mL) was added to the residue. Conc. HCl was added dropwise to precipitate the desired product; 1.05 g (100% yield).
<Example 10>
[(S) -trans]-[1- (but-2-enyl-{[(1-phenyl-cyclobutylmethyl) -carbamoyl] -methyl} -carbamoyl) -2- (3H-imidazole-4 -Yl) -ethyl] -carbamic acid benzyl ester
Step 1: Methyl 2-[(E) -2-butenylamino] acetate
(E) -2-buten-1-amine.HCl (5.37 g, 49.9 mmol) in acetonitrile (100 mL) [Chem. Ber., 1984; 117: 1250] The suspension was treated with methyl bromoacetate (4.72 mL, 49.9 mmol) and Et ₃ N (14.0 mL, 99.8 mmol) and stirred at room temperature for 1 hour. The suspension was then heated to reflux overnight. The solution developed at reflux temperature. After cooling, the precipitated Et ₃ N.HCl was filtered off and the solvent was removed under reduced pressure to leave 5.0 g of crude product. Chromatography on silica gel eluted with CHCl ₃ / MeOH (98: 2) to afford 1.41 g (19.8% yield) of the pure product as an oil.
Step 2: Methyl 2- [2-[(benzyloxy) carbonyl] amino-3- (1-trityl-1H-5-imidazolyl) propanoyl] [(E) -2-butenyl] amino acetate
A solution of methyl 2-[(E) -2-butenylamino] acetate (product of step 1) (0.6 g, 4.2 mmol) in CH ₂ Cl (50 mL) was cooled on ice and Z-His (Trt) (2.23 g , 4.2 mmol), diisopropylethylamine (2.2 mL, 12.6 mmol) and PyBOP (2.2 g, 4.2 mmol). After stirring for 15 minutes at 0 ° C., the solution was stirred for 4 days at room temperature. After removal of solvent under reduced pressure the residue was taken up with EtOAc, washed three times with H ₂ O and then with saturated NaCl. Drying with MgSO ₄ and removing the solvent under reduced pressure gave 4.36 g of crude product. Chromatography on silica gel eluted with CHCl ₃ / MeOH (98: 2) to give 2.23 g (81.1% yield) of the pure product as a solid foam.
MS, m / z 657 (M + H ⁺ ).
Step 3: 2- [2-[(benzyloxy) carbonyl] amino-3- (1-trityl-1H-5-imidazolyl) propanoyl] [(E) -2-butenyl] aminoacetic acid
A solution of ester (compound of step 2) (2.23 g, 3.4 mmol) in MeOH / dioxane (20 mL and 15 mL, respectively) was treated with 2N NaOH (7.0 mL, 14.0 mmol) and stirred at room temperature for 0.5 h. After addition of 2N HCl (7.0 mL, 14.0 mmol) the mixture was evaporated to give a solid. It was mixed with EtOAc / THF and filtered to remove NaCl. The solvent was removed under reduced pressure to give 2.06 g (94.5% yield) of the product as a solid foam.
MS, m / z 643 (M + H ⁺ ).
Step 4: benzyl N-2-[(E) -2-butenyl (2-oxo-2-[(1-phenylcyclobutyl) methyl] aminoethyl) amino] -2-oxo-1-[(1- Trityl-1H-4-imidazolyl) methyl] ethyl carbamate
The compound of step 3 (1 g, 1.6 mmol) was dissolved in methylene chloride (50 mL). HOBt (0.29 g, 2.1 mmol) was added followed by 0.5 M DCC / DCM (3.8 mL, 1.9 mmol), C- (1-phenyl-cyclobutyl) -methylamine hydrochloric acid (0.37 g, 1.9 mmol) and DIEA (0.61 mL) , 3.5 mmol) was added. The reaction was stirred for 2 days at room temperature under a nitrogen environment. The solution was filtered and the filtrate was concentrated under reduced pressure. The residue was taken up in ethyl acetate and the organic solution was washed twice with 2N HCl and with 1N NaHCO ₃ and brine, dried over MgSO ₄ , filtered and concentrated. Chromatography on silica gel eluted with CHCl ₃ / MeOH (95: 5) to afford 0.97 g (77% yield) of the pure product.
Step 5: [(S) -Trans]-[1- (But-2-enyl-{[(1-phenyl-cyclobutylmethyl) -carbamoyl] -methyl} -carbamoyl) -2- (3H-already Dazol-4-yl) -ethyl] -carbamic acid benzyl ester
The compound of step 4 (0.97 g, 1.2 mmol) was refluxed with glacial acetic acid (20 mL) and water (5 mL) for 40 minutes to afford the title compound. The reaction mixture was cooled down and concentrated under reduced pressure. The residue was taken up in ethyl acetate and the organic solution was washed four times with saturated NaHCO ₃ , twice with brine, dried over MgSO ₄ , filtered and concentrated. Chromatography on silica gel eluted with CHCl ₃ / MeOH (0-6% CH ₃ OH in CHCl ₃ ) to afford 0.43 g (66% yield) of the pure product.
Analytical calcd. For C ₃₁ H ₃₇ N ₅ O ₄ : 0.25 CHCl ₃ :
C, 65.45; H, 6.55; N, 12.21.
Found: C, 65.59; H, 6. 61; N, 12.20.
MS-APCI: Calcd for M + 1: 544.7; Found: 544.3.
<Example 11>
(S)-[2- (3H-imidazol-4-yl) -1-({[(1-phenyl-cyclobutylmethyl) -carbamoyl] -methyl} -propyl-carbamoyl) -ethyl] -carr Chest Acid Benzyl Ester
Step 1 can be carried out as follows and the title compound can be prepared according to Example 10. The title compound was obtained as a white foam; 0.25 g (66% yield).
C ₃₀ H ₃₇ N ₅ O ₄ : Analytical calculations of 0.10 CHCl ₃ :
C, 66.51; H, 6.88; N, 12.88.
Found: C, 65.52; H, 6. 84; N, 13.11.
MS-APCI: Calcd for M + 1: 532.7; Found: 532.2.
Step 1: Methyl 2- (propylamino) acetate
A suspension of glycine methyl ester.HCl (5.0 g, 39.8 mmol) in acetonitrile (100 mL) was treated with propyl bromide (3.7 mL, 39.8 mmol) and diisopropylethylamine (13.9 mL, 79.6 mmol). After stirring for 1 hour at room temperature the mixture was heated to reflux overnight. The solution developed at reflux temperature. The solvent was removed under reduced pressure and the residue was treated with Et ₂ O / EtOAc. The solvent was filtered off under pressure leaving 1.13 g of crude product. Chromatography on silica gel eluted with CHCl ₃ / MeOH (98: 2) to give 0.53 g (10.2% yield) of the product as a yellow oil.
MS, m / z 132 (M + H ⁺ ).
<Example 12>
Benzyl N-((1S) -1- (1H-4-imidazolylmethyl) -2-oxo-2- (2-oxo-2-[(1-phenylcyclobutyl) methyl] aminoethyl) [(1R ) -1-phenylethyl] aminoethyl) carbamate
The title compound can be prepared by adding the following changes to Example 10: In step 1, replace (E) -2-buten-1-amine-HCl with R-α-methylbenzylamine; In step 2, replace with HATU instead of PyBOP; In step 3, LiOH: H ₂ O in THF was used instead of 2N NaOH in methanol / dioxane. The title compound was obtained as white foam; 0.51 g (66% yield).
Analytical calculation of C ₃₅ H ₃₉ N ₅ O ₄ 0.35 CH ₂ Cl ₂ :
C, 68.10; H, 6. 42; N, 11.23.
Found: C, 68.25; H, 6. 46; N, 11.23.
MS-APCI: Calcd for M + 1: 594.7; Found: 594.2.
Example 13
(S)-[1-((1,1-Dimethyl-2-phenyl-ethyl)-{[(1-phenyl-cyclobutylmethyl) -carbamoyl] -methyl} -carbamoyl) -2- (3H- Imidazol-4-yl) -ethyl] -carbamic acid benzyl ester
The title compound can be prepared by adding the following changes to Example 10: In step 1, β, β-dimethylphenethylamine hydrochloric acid instead of (E) -2-butene-1-amine-HCl (step A below) Replace with; In step 2, using HATU and HOAt instead of PyBOP; In step 4, using PyBOP instead of DCC / HOBt; In step 5 95% TFA in DCM was used instead of glacial acetic acid in water. The title compound was obtained as white foam; 0.121 g (12% yield).
Analytical calculation of C ₃₇ H ₄₂ N ₅ O ₄ 0.33 CH ₂ Cl ₂ :
C, 69.11; H, 6.63; N, 10.79.
Found: C, 69.10; H, 6.91; N, 11.00.
MS-APCI: Calcd for M + 1: 621.8; Found: 622.2.
Step A: β, β-dimethylphenethylamine hydrochloride
Sodium hydride (60% in oil) (17 g, 0.43 mol) was suspended in THF (150 mL) and cooled to 0 ° C. under nitrogen. Benzyl cyanide (22.2 g, 0.19 mol) in THF (30 mL) was added dropwise and the reaction was stirred for 1 hour. Methane iodide (24.9 mL, 0.4 mol) in THF (20 mL) was added dropwise at 0 ° C. The reaction was stirred overnight at room temperature under nitrogen. The solution was filtered and the filtrate was removed in vacuo. The residue was taken up with ethyl acetate (100 mL) and washed three times with 10% NaHSO ₃ , and saturated NaHCO ₃ , brine, dried over MgSO ₄ and concentrated; 22.74 g (92% yield).
The product was reduced to Raney nickel in methanol / NH ₃ . The catalyst was removed and washed with methanol. The filtrate was concentrated and diethyl ether (100 mL) was added to the residue. Concentrated hydrochloric acid was added dropwise to precipitate the desired product; 24.8 g (86% yield).
PFT inhibitory activity
Protein of the compounds of the invention: farnesyl transferase (PFT) or farnesyl protein transferase (FPT) inhibitory activity was assayed in HEPES buffer (pH 7.4) containing 5 mM potassium phosphate and 20 μM ZnCl ₂ . The solution also contained 5 mM DTT (dithiothritol), 5 mM MgCl ₂ and 0.1% PEG 8000. Assays were performed in 96 well plates (Wallec), using solutions containing various concentrations of compounds of the invention in 100% DMSO (dimethylsulfoxide). Both substrates, radiolabeled farnesyl pyrophosphate ([1- ³ H], characteristic activity 15-30 Ci / mmol, final concentration 134 nM) and (biotinyl) -Ahe-Thr-Lys-Cys-Val-Ile- Met ([3aS [3a alpha, 4 beta, 6a alpha] -hexahydro-2-oxo-1H-thieno [3,4-d] imidazole-5-pentanoic acid]-[7-aminoheptanoic acid]- Thr-Lys-Cys-Val-Ile-Met) (Ahe is 7-aminoheptanoic acid, Thr is threonine, Lys is lysine, Cys is cysteine, Val is valine, Ile isoleucine, and Met is methionine) (final) The enzyme reaction was started by adding SF9 affinity purified mouse panesyl protein transferase to the concentration of 0.2 μM). After 30 min incubation at 30 ° C., stop buffer containing 1.5 M magnesium acetate, 0.2 MH ₃ PO ₄ , 0.5% BSA (bovine serum albumin) and 1.3 mg / mL of strepavidin beads (Amersham) The solution was terminated by diluting the solution 2.5 times). After the plate was fixed at room temperature for 30 minutes, radioactivity was quantified by a microbeta counter (model 1450, Wallec). The assay was also performed in the absence of 5 mM potassium phosphate.
Gel transfer assay
Various concentrations of panesylation inhibitor are added 24 hours after planting 2 × 10 ⁶ las-transformed cells per treatment condition. After 18 hours of incubation, several protease inhibitors (PMSF) in phosphate-buffered saline at pH 7.4 containing 1% Triton X-100, 0.5% sodium dioxycholate and 0.1% SDS (sodium dodecyl sulfate) (Phenylmethylsulfonylfluoride), antipine, leupeptin, pepstatin A and aprotinin total 1 μg / mL) are lysed. 3 μg of vH-Ras Ab-2 (Y13-259 antibody from Oncogene Science) is added to irradiate Ras protein from the supernatant. After overnight immunoprecipitation, 30 μL of 50% Protein G-Sepharose slurry (Pharmacia) is added and incubated for 45 minutes. The pellet is resuspended in 2 × Tris-glycine loading buffer (Novex) containing 5% β-mercaptoethanol and boiled for 5 minutes prior to electrophoresis on a 14% Tris-glycine SDS gel. The protein is transferred to nitrocellulose membrane using Western transfer technique and then blocked in blocking buffer. After incubation with the primary antibody (Pan-Las Ab-2 from Oncogene Science) overnight, the Ras protein is detected using an antimouse HRP (horse radish peroxidase) conjugate secondary antibody (Amersham). The blot is developed using the ECL (Enhanced Chemiluminescence) technique (Amersham).
Clonogenicity Assay (6-well Plate)
Just before installing the actual test:
1. Form 1.5% bacterium agar in Milli-Q water and autoclave.
2. Make 500 mL of 2X DMEM-HG without phenol red by combining the following:
1 bottle of DMEM base powder (Sigma D-5030)
4.5 g glucose
3.7 g of sodium bicarbonate
0.11 g sodium pyruvate
20mL 200mM L-Glutamine (Sigma G-7513)
1 mL pen-strep (GibcoBRL No. 15140-023)
Adjust pH to 7.1 with HCl; Sterilize the filter.
........................................ .....................
1. Install a temporary bath in the hood (the thermometer is plugged into a beaker of water and placed on a hot plate). The water temperature is maintained between 37 ° C. and 43 ° C.
2. Autoclave 1.5% Bakto agar at high temperature for about 2 minutes or until completely dissolved. Then cool it shortly before use. (Place bottle on hot plate to prevent resolidification.)
3. Bottom layer (0.6% baby) Top layer (0.3% baby)
20% Calf Serum 20% Calf Serum
40% 2X DMEM 50% 2X DMEM
40% Bacto Agar (1.5%) 20% Bacto Agar (1.5%)
10% sterile H ₂ O x μL cell suspension
(to = 5000 cells / well) (H61 cells: NIH
Transgenic 3T3 H-Ras cells)
Depending on the volume of each layer required, use 50 mL conical tubes or 200 mL turbine tubes that can float in the bath.
4. Add 1 mL bottom layer agar / medium to each well: transfer 1 mL warm agar / medium to the well; Then use the tip of the pipette to spread the agar / medium to cover the bottom completely. Repeat for the next well. The last mL in the pipette generates bubbles and is not added to the wells.
5. Allow the plate to settle for about 5 minutes at room temperature to solidify the bottom layer.
6. Mark a sterile Falcon 2054 (12 x 75 mm) tube and add the appropriate volume of drug solution to it.
7. Aliquot 4 μL of DMSO or drug solution per 1 mL of agar / medium into a suitable tube; Agar / medium / cells are then added to each tube. Always add 1 mL more than is actually needed. Mix up and down with a pipette (soft); Then transfer 1 mL to the center of each well. The top layer will be less viscous and will therefore generally spread to the bottom layer without much effort. If necessary, gently rotate the face of the plate to spread the top layer evenly to the bottom layer.
8. Allow plate to solidify for 5 or 10 minutes at room temperature and place in 5% CO ₂ , 37 ° C incubator.
9. On day 13, add 0.5 mL of INT (1 mg / mL tetrazolium in milli-QH ₂ O, sterile strainer) and return the plate to the incubator.
10. Count colonies.
The data in the table below show the farnesyl protein transferase inhibitory activity, activity in gel transfer assays and activity in clonality assays of the compounds of the present invention.
Example numberPFT inhibitory activityGel transfer (μM) MED ^* Clonogenicity Analysis IC ₅₀ (μM) IC ₅₀ (μM) HEPESIC ₅₀ (μM) HEPES and 5mM K ₃ PO ₄1234567891011120.0750.150.140.260.320.260.0140.0687.0 <0.001 <0.0010.0020.0060.0060.00170.00540.0180.00290.0030.0150.180.360.330.340.020.020.0020.010.20.0020.010.01> 0.2≤0.05≤0.05≤0.050.0470.180.14NT0.320.21NTNTNTNTNTNT
* M.E.D. is the minimum effective amount to observe the inhibition of las panesylation
# NT = not tested
In vivo activity
Female NCR-NU mice were randomized and inoculated with trocar pieces of H61 xenografts obtained from donor animals on day 0 of the experiment. H61 cells are NIH3T3 cells transformed by transfection with mutant human H-ras. Animals are then rearranged and included in the treatment group. Mice with tumors were then treated via subcutaneous (SC) injection or peritoneal (IP) injection in Example 1 at various doses every 12 hours starting on day 1 and every 14 days. Tumors were measured on the orthogonal two-dimensional plane side by side several times each week during the experiment. Standard methods to measure the tumor load by caliper measurement by the (mg = axb ^2/2, the equation a and b are measured ¹ of major axis and minor axis of tumor, respectively) in mg units. Tumor growth inhibition was assessed on the last day of treatment (14). Example 1 in this assay significantly inhibited tumor growth. When 96 mg / kg / subcutaneous injection was administered, the median tumor load of the treatment group was 672 mg compared to the median load of 2508 mg for the excipient treatment control group, indicating 73% inhibition of tumor growth. For mice administered Example 1 37 or 23 mg / kg / peritoneal injection, no tumor appeared at the site of inoculation indicating complete (100%) inhibition of tumor growth.

权利要求:
Claims (23)
[1" claim-type="Currently amended] Compounds of formula I and their pharmaceutically acceptable salts, esters, amides and prodrugs.
<Formula I>

In the above formula
R ^Q is

ego;
x is 0 or 1;
Each R ¹⁴ is independently hydrogen or C ₁ -C ₆ alkyl;
A is -COR ^a , -CO ₂ R ^{a '} , -CONHR ^a' , -CSR ^a , , , -C (S) OR ^{a '} , -C (S) NHR ^a' , -SO ₂ R ^a or -CONR ^a R ^{a ''} ;
R ^a , R ^{a '} and R ^a'' are independently C ₁ -C ₆ alkyl,-(CR ¹⁴ R ¹⁴ ) _m -cycloalkyl,-(CR ¹⁴ R ¹⁴ ) _m -aryl, or-(CR ¹⁴ R ¹⁴ ) _m -heteroaryl;
Each m is independently 0-3;
R ¹ , R ² and R ⁴ are independently hydrogen or C ₁ -C ₆ alkyl;
R ³ is , C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl,-(CR ¹⁴ R ¹⁴ ) _m -naphthyl,-(CH ₂ ) _v CO ₂ R ¹⁴ ,-(CH ₂ ) _t NR ¹⁴ R ¹⁴ , -(CH ₂ ) _v -OC ₁ -C ₆ alkyl,-(CH ₂ ) _t -OH,-(CH ₂ ) _t -morpholino, , ,-(CR ¹⁴ R ¹⁴ ) _m- (phenyl substituted with R ^b ) or-(CR ¹⁴ R ¹⁴ ) _m- (heteroaryl substituted with R ^b );
t is 2 to 6;
v is 1 to 6;
R ^b is —O-phenyl, —O-benzyl, halogen, C ₁ -C ₆ alkyl, hydrogen, —OC ₁ -C ₆ alkyl, -NH ₂ , -NHR ^a , -NR ^a R ^{a '} , , , -OH, , , , , -O (CH ₂ ) _y NR ^a R ^{a '} , -CF ₃ , -NO ₂ , , , -CN, -OPO ₃ H ₂ , -CH ₂ PO ₃ H ₂ , , -N ₃ , -CF ₂ CF ₃ , -SO ₂ R ^a , -SO ₂ NR ^a R ^{a '} , -CHO, -OCOCH ₃ , -O (CH ₂ ) _m -aryl, -O (CH ₂ ) _m -Cycloalkyl,-(CH ₂ ) _m -aryl,-(CH ₂ ) _m -cycloalkyl,-(CH ₂ ) _m -heteroaryl, or -CH = CHC ₆ H ₅ , or -O (CH ₂ ) _m Heteroaryl;
y is 2 or 3;
R ⁵ is

ego;
Each n is independently 2, 3 or 4;
R ⁱ , R ^g and R ^h are independently hydrogen, halogen, -OC ₁ -C ₆ alkyl, C ₁ -C ₆ alkyl, -CN, -OPO ₃ H ₂ , -CH ₂ PO ₃ H ₂ , -O- Phenyl, -O-benzyl, , , , , -O (CH ₂ ) _y NR ^a R ^{a '} , -NH ₂ , -NHR ^a , -NR ^a R ^a' , , , -OH, CF ₃ , -NO ₂ , , , , -N ₃ , -CF ₂ CF ₃ , -SO ₂ R ^a , -SO ₂ NR ^a R ^{a '} , -CHO or -OCOCH ₃ ;
R ^c and R ^d are independently C ₁ -C ₆ alkyl, — (CH ₂ ) _m -cycloalkyl or hydrogen.
[2" claim-type="Currently amended] The compound of claim 1, wherein R ¹ is hydrogen, R ² is hydrogen, R ⁴ is hydrogen, R ¹⁴ is hydrogen or methyl,
A is

Phosphorus compounds.
[3" claim-type="Currently amended] The compound of claim 1, wherein R ³ , C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl,-(CH ₂ ) _m- (phenyl substituted with R ^b ) or-(CH ₂ ) _m- (heteroaryl substituted with R ^b );
R ¹ is hydrogen, R ² is hydrogen, R ⁴ is hydrogen, and R ¹⁴ is hydrogen or methyl.
[4" claim-type="Currently amended] The compound of claim 1 wherein R ⁵ is

Phosphorus compounds.
[5" claim-type="Currently amended] Compounds of formula II and their pharmaceutically acceptable salts, esters, amides and prodrugs.
<Formula II>

In the above formula
R ⁶ is -O-benzyl, , -NH-benzyl, -N (C ₁ -C ₆ alkyl) -benzyl or -SCH ₂ -phenyl;
R ⁸ is hydrogen, halogen, C ₁ -C ₆ alkyl, —O-benzyl, —OCH ₂ -pyridyl, —OC ₁ -C ₆ alkyl, —CF ₃ , —OH or —phenyl;
R ¹⁰ and R ¹³ are independently hydrogen or C ₁ -C ₆ alkyl;
Each n is independently 2, 3 or 4;
R ¹² is ego;
R ¹⁴ is hydrogen or methyl;
R^j, R^kAnd R^lIs independently hydrogen, -NH₂, -NHR^a, Halogen, -OC_One-C₆Alkyl or -C_One-C₆Alkyl.
[6" claim-type="Currently amended] Compounds of formula III and their pharmaceutically acceptable salts, esters, amides and prodrugs.
<Formula III>

In the above formula
Each n is 2, 3 or 4;
X is NH, O, or -NCH ₃ ;
R ¹⁵ is —O-benzyl, —CF ₃ , hydrogen, halogen, —OH, —phenyl, —C ₁ -C ₆ alkyl, —O—CH ₂ -pyridyl or —OC ₁ -C ₆ alkyl.
[7" claim-type="Currently amended] Compound of formula IV.
<Formula IV>

In the above formula
X is NH, O, or -NCH ₃ ;
R ¹⁴ is hydrogen or C ₁ -C ₆ alkyl;
R ^m is C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl,-(CH ₂ ) _t NR ¹⁴ R ¹⁴ ,-(CH ₂ ) _v -OC ₁ -C ₆ alkyl,-(CH ₂ ) _t- OH,-(CH ₂ ) _t -morpholino, , ,-(CH ₂ ) _v -CO ₂ R ¹⁴ or ego;
n is 2, 3 or 4;
m is 0 to 3;
t is 2 to 6;
v is 1-6.
[8" claim-type="Currently amended] A pharmaceutically acceptable composition comprising the compound of claim 1.
[9" claim-type="Currently amended] A pharmaceutically acceptable composition comprising the compound of claim 5.
[10" claim-type="Currently amended] A pharmaceutically acceptable composition comprising the compound of claim 6.
[11" claim-type="Currently amended] A method of treating or preventing restenosis comprising administering a therapeutically effective amount of a compound of claim 1 to a patient with or at risk of developing restenosis.
[12" claim-type="Currently amended] A method of treating or preventing restenosis comprising administering a therapeutically effective amount of a compound of claim 5 to a patient with or at risk of developing restenosis.
[13" claim-type="Currently amended] A method of treating or preventing restenosis comprising administering a therapeutically effective amount of a compound of claim 6 to a patient with or at risk of developing restenosis.
[14" claim-type="Currently amended] A method of treating cancer, comprising administering to a patient with cancer a therapeutically effective amount of the compound of claim 1.
[15" claim-type="Currently amended] A method of treating cancer, comprising administering to a patient with cancer a therapeutically effective amount of the compound of claim 5.
[16" claim-type="Currently amended] A method of treating cancer, comprising administering a therapeutically effective amount of the compound of claim 6 to a patient with cancer.
[17" claim-type="Currently amended] The following compounds:
(S)-[1-((4-benzyloxy-benzyl)-{[(1-phenyl-cyclobutylmethyl) -carbamoyl] -methyl} -carbamoyl) -2- (1H-imidazole-4- Yl) -ethyl] -carbamic acid benzyl ester;
(S)-[1-((4-benzyloxy-benzyl)-{[(1-phenyl-cyclopropylmethyl) -carbamoyl] -methyl} -carbamoyl) -2- (1H-imidazole-4- Yl) -ethyl] -carbamic acid benzyl ester;
(S)-[1-((4-Benzyloxy-benzyl)-{[(1-phenyl-cyclopentylmethyl) -carbamoyl] -methyl} -carbamoyl) -2- (1H-imidazole-4- Yl) -ethyl] -carbamic acid benzyl ester;
(S)-[1-((4-phenyl-benzyl)-{[(1-phenyl-cyclobutylmethyl) -carbamoyl] -methyl} -carbamoyl) -2- (1H-imidazol-4-yl ) -Ethyl] -carbamic acid benzyl ester;
(S)-[1-((4-methoxy-benzyl)-{[(1-phenyl-cyclobutylmethyl) -carbamoyl] -methyl} -carbamoyl) -2- (1H-imidazole-4- Yl) -ethyl] -carbamic acid benzyl ester;
(S)-[1-((4-Methyl-benzyl)-{[(1-phenyl-cyclobutylmethyl) -carbamoyl] -methyl} -carbamoyl) -2- (1H-imidazol-4-yl ) -Ethyl] -carbamic acid benzyl ester;
(S) -N- (4-benzyloxy-benzyl) -2- (3-benzyl-ureido) -3- (1H-imidazol-4-yl) -N-{[(1-phenyl-cyclobutyl Methyl) -carbamoyl] -methyl} -propionamide;
(S) -2- (3-benzyl-3-methyl-ureido) -N- (4-benzyloxy-benzyl) -3- (1H-imidazol-4-yl) -N-{[(1- Phenyl-cyclobutylmethyl) -carbamoyl] -methyl} -propionamide;
(S)-[1-((4-benzyloxy-benzyl)-{[(1-phenyl-cyclobutylmethyl) -carbamoyl] -methyl} -carbamoyl) -2- (1H-imidazole-4- Yl) -ethyl] -thiocarbamic acid S-benzyl ester;
(S)-(2- (1H-imidazol-4-yl) -1-{{[(1-phenyl-cyclobutylmethyl) -carbamoyl] -methyl}-[4- (pyridin-2-ylmethoxy ) -Benzyl] -carbamoyl} -ethyl) -carbamic acid benzyl ester;
(S)-(1-((cyclohexyl-methyl)-{[(1-phenyl-cyclobutylmethyl) -carbamoyl] -methyl} -carbamoyl) -2- (1H-imidazol-4-yl) -Ethyl] -carbamic acid benzyl ester; and
(S)-(1-((Isobutyl)-{[(1-phenyl-cyclobutylmethyl) -carbamoyl] -methyl} -carbamoyl) -2- (1H-imidazol-4-yl) -ethyl ] -Carbamic acid benzyl ester.
[18" claim-type="Currently amended] The following compounds:
(S) -2- (3-benzyl-3-methyl-ureido) -3- (1H-imidazol-4-yl) -N- (4-methyl-benzyl) -N-{[(1-phenyl -Cyclobutylmethyl) -carbamoyl] -methyl} -propionamide;
(S)-[1-[(4-Benzyloxy-benzyl)-({[1- (2, 6-dichloro-phenyl) -cyclobutylmethyl] -carbamoyl} -methyl) -carbamoyl] -2- (1H-imidazol-4-yl) -ethyl] -carbamic acid benzyl ester;
[(S) -trans]-[1- (but-2-enyl-{[(1-phenyl-cyclobutylmethyl) -carbamoyl] -methyl} -carbamoyl) -2- (3H-imidazole-4 -Yl) -ethyl] -carbamic acid benzyl ester;
(S)-[2- (3H-imidazol-4-yl) -1-({[(1-phenyl-cyclobutylmethyl) -carbamoyl] -methyl} -propyl-carbamoyl) -ethyl] -carr Chest acid benzyl ester;
Benzyl N-((1S) -1- (1H-4-imidazolylmethyl) -2-oxo-2- (2-oxo-2-[(1-phenylcyclobutyl) methyl] -aminoethyl) [( 1S) -1-phenylethyl] aminoethyl) carbamate; And
(S)-[1-((1,1-Dimethyl-2-phenylethyl)-{[(1-phenyl-cyclobutylmethyl) -carbamoyl] -methyl} -carbamoyl) -2- (3H-im already Dazol-4-yl) -ethyl] -carbamic acid benzyl ester.
[19" claim-type="Currently amended] The following compounds:
Benzyl N-[(1S) -2-[(2-hydroxyethyl) (2-oxo-2-[(1-phenylcyclobutyl) methyl] aminoethyl) amino] -1- (1H-4-imida Zolylmethyl) -2-oxoethyl] carbamate;
3-[[(2S) -2-[(benzyloxy) carbonyl] amino-3- (1H-4-imidazolyl) propanoyl] (2-oxo-2-[(1-phenylcyclobutyl) Methyl] aminoethyl) amino] propanoic acid;
Methyl 3-[[(2S) -2-[(benzyloxy) carbonyl] amino-3- (1H-4-imidazolyl) propanoyl] (2-oxo-2-[(1-phenylcyclobutyl ) Methyl] aminoethyl) amino] propanoate;
Benzyl N-[(1S) -2-[(2-aminoethyl) (2-oxo-2-[(1-phenylcyclobutyl) methyl] aminoethyl) amino] -1- (1H-4-imidazolyl Methyl) -2-oxoethyl] carbamate;
Benzyl N- (1S) -1- (1H-4-imidazolylmethyl) -2-[[2- (methylamino) ethyl] (2-oxo-2-[(1-phenylcyclobutyl) methyl] amino Ethyl) amino] -2-oxoethylcarbamate;
Benzyl N- (1S) -1- (1H-4-imidazolylmethyl) -2-[(2- (methoxyethyl) (2-oxo-2-[(1-phenylcyclobutyl) methyl] aminoethyl ) Amino] -2-oxoethylcarbamate;
Benzyl N- (1S) -1- (1H-4-imidazolylmethyl) -2-[(2- (morpholinoethyl) (2-oxo-2-[(1-phenylcyclobutyl) methyl] amino Ethyl) amino] -2-oxoethylcarbamate;
Benzyl N- (1S) -1- (1H-4-imidazolylmethyl) -2-[(2- (methyl-2-phenylpropyl) (2-oxo-2-[(1-phenylcyclobutyl) methyl ] Aminoethyl) amino] -2-oxoethyl carbamate; and
1-phenylethyl N-[(1S) -2-[[4- (benzyloxy) -1,5-cyclohexadienyl] methyl (2-oxo-2-[(1-phenylcyclobutyl) methyl] amino Ethyl) amino] -1- (1H-4-imidazolylmethyl) -2-oxoethyl] carbamate.
[20" claim-type="Currently amended] The method of claim 13, wherein the cancer is lung cancer, colon cancer, breast cancer, pancreatic cancer, thyroid cancer, or bladder cancer.
[21" claim-type="Currently amended] A method of treating psoriasis comprising administering to a patient with psoriasis a therapeutically effective amount of the compound of claim 1.
[22" claim-type="Currently amended] A method of treating psoriasis comprising administering to a patient with psoriasis a therapeutically effective amount of the compound of claim 5.
[23" claim-type="Currently amended] A method of treating psoriasis comprising administering to a patient with psoriasis a therapeutically effective amount of the compound of claim 6.

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引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
1996-12-17|Priority to US3366196P

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1996-12-17|Priority to US60/033,661

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1997-08-22|Priority to US5683197P

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1997-08-22|Priority to US60/056,831

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1997-12-16|Application filed by 로즈 암스트롱, 크리스틴 에이. 트러트웨인, 워너-램버트 캄파니

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2000-09-25|Publication of KR20000057601A

优先权:

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申请号 | 申请日 | 专利标题

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US3366196P| true| 1996-12-17|1996-12-17|

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US60/033,661|1996-12-17|

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US5683197P| true| 1997-08-22|1997-08-22|

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US60/056,831|1997-08-22|