KR19980072748A - Trans-pyrrolidine derivatives useful as panesyl transferase inhibitors - Google Patents

Abstract

The present invention relates to novel trans-pyrrolidine derivatives represented by the following formula (1), pharmaceutically acceptable salts thereof, and methods for their preparation.

[Formula 1]

(In Formula 1, R ₁ , R ₂ and R ₃ are as defined in the specification.)

The trans-pyrrolidine derivatives and their pharmaceutically acceptable salts can act usefully as anticancer agents because they act to inhibit farnesyl transferase.

Description

Trans-pyrrolidine derivatives useful as panesyl transferase inhibitors

The present invention relates to trans-pyrrolidine derivatives and their pharmaceutically acceptable salts, their preparation and their use as panesyl transferase inhibitors.

More specifically, the present invention relates to a trans-pyrrolidine derivative and a pharmaceutically acceptable salt thereof, and a method for preparing the same, wherein the trans-pyrrolidine derivative of the present invention It can be usefully used as an anticancer agent.

Panesyl transferase is an enzyme that transfers panesyl groups to Ras protein, and the compound of the present invention inhibits the action of Ras protein by inhibiting the action of panesyl transferase.

Ras protein is a 21 kDa protein that plays an important role in cell growth and differentiation. It binds to guanine nucleotides, hydrolyses guanosine triphosphate (GTP) to guanosine diphosphate (GDP), or phosphorylates GDP to GTP. It works. Since GTP activates G protein and GDP inhibits G protein, Ras protein acts as a molecular switch that regulates the cellular signaling system by G protein (Bourne, H. R, Sanders, D. A, McCormick, F., Nature, 1991, 349, 117).

Ras proteins are produced from three ras genes in mammalian cells and include the K-Ras-4B protein of 188 amino acids or the H-Ras, K-Ras4A and N-Ras proteins of 189 amino acids. The amino acids at positions 12, 13, and 61 in these proteins are in close proximity to the phosphate groups of GTP, and these amino acid residues affect the spatial position of the water molecules involved in the process of GTP hydrolysis, thus affecting the GTPase enzyme activity of the Ras protein. Plays an important role. Specifically, some cancer cells generated in the human body are observed to be mutated at the amino acid position. When this mutation inhibits the Ras protein-specific GTPase enzyme activity, the GTP binding state is continued and abnormal growth signals are continuously transmitted. In addition, it has been reported that carcinogenicity is caused by abnormalities in these signaling systems. Indeed, these carcinogenic ras genes are known to be closely associated with pancreatic cancer, bladder cancer, lung cancer and skin cancer (Bos, JL, Cancer Res., 49, 4682, 1989).

In order for Ras protein to be biologically active, it must be attached to the cell membrane, and in order for Ras protein to be attached to the cell membrane, it must first be able to easily bind to the lipid layer in the cell membrane. Ras proteins are hydrophobic through several stages of binding, specifically the process by Ras farnesyltransferase involved in panesylation, three amino acids present at the carboxy terminus of Ras protein. AAX peptide cleavage process, methyl transferase and palmitoyl transferase process, and the like, the carboxy terminal of the Ras protein is modified by this process. The first of these processes, panesylation, is carried out by panenyl transferase, in which a peptide consisting of four amino acids, CA1A2X, present at the carboxy terminus of the Ras protein is used as a substrate. Where A1 and A2 are aliphatic amino acids with no electrical load and X is methionine, alanine and serine. This panesylation reaction takes place at the cysteine site to form sulfur ether bonds, especially for H-Ras and N-Ras proteins, which also cause palmitoylation of another cysteine near its carboxy terminus. As a result of this panesylation, the Ras protein is increased in hydrophobicity and can be attached to the cell membrane, and the panesylated Ras protein is methylated again when the peptide of the three amino acids AAX at its carboxy terminus is removed by a cleavage enzyme. It is known that the farnesyl groups of proteins readily bind to lipid layers or other receptors in cell membranes. Indeed, although all of the above modification steps are necessary for optimal attachment of Ras proteins to cell membranes, it is reported that panesylation alone is sufficient for Ras protein activity. Therefore, blocking the panesylation process effectively inhibits the carcinogenicity of Ras proteins caused by mutations, and thus, studies to inhibit panesylation have been actively conducted (Buss, JE et al., Chemistry Biology, 2, 787, 1995).

Previous studies have shown that inhibition of farnesyl transferase not only inhibits cell growth transformed with Ras protein but also improves cell traits modified by Ras protein. It has been shown to selectively inhibit the response of oncogenic Ras proteins by intracellular prenyl groups [Kokl, NE et al., Proc. Natl. Acad. Sci. USA, 91: 9141 (1994); Kokl, N. E. et al., Nature Medicine, 1: 792 (1995).

Inhibitors of the farnesyl transferase, which combines two substrates, the farnesyl group and the Ras protein to generate a reactant, can be classified into three types. First, a compound capable of competitively inhibiting the farnesyl group (FPP), second, a compound that inhibits the C-terminus of the Ras protein, and a farnesyl transferase mimic the activation stage of the catalytic reaction using two substrates. It is to apply a stable compound as an inhibitor.

Most of the inhibitors studied so far are related to the CAAX motif, which mediates the introduction of the prenyl group at the C-terminus of the Ras protein, and applies a competitive inhibitory mechanism of the farnesyl transferase to the Ras protein substrate. For example, Kokl, N. E. et al. Studied peptide variants containing cysteine thiol groups that mimic CAAX and inhibitors that improved them [US Pat. Nos. 5,141,851; Kokl, N. E. et al., Science 260: 1934 (1993); PCT / US95 / 12224, Graham et al., Septi S. M., et al., Studied panesyl transferase inhibitors in which the skeletal structure of peptides was modified with phenyl groups [Sebti S. M. et al., J. Biol. Chem. 270: 26802 (1995). In addition, a variant using a benzodiazepine as a turn simulation structure of a peptide has been reported in the psychotropic pharmaceutical framework [James, GL et al., Science 260: 1937 (1993)], and tricycline deviated from the peptide structure. Inhibitors based on organic compounds have been studied [Bishop WR et al., J. Biol. Chem. 270: 30611 (1995).

On the other hand, studies of inhibitors that mimic the catalysis of panesyl transferase suggest that Poulter CD et al suggest that the mechanism of action of panesyl transferase to transfer prenyl groups is electrophilic displacement. , CD et al., Poc. Natl. Acad. Sci. USA], focusing on the response that requires a positive load in the transition state, we studied a new type of inhibitor that linked the positive load of the transition state to the prenyl group [Poulter C. D. et al., J. Am. Chem. Soc. 118: 8761 (1996).

As a result of efforts to develop new farnesyl transferase inhibitors, the present inventors have completed the present invention by preparing a trans-pyrrolidine derivative having excellent panesyl transferase inhibitory ability.

It is an object of the present invention to provide a novel trans-pyrrolidine derivative which acts to inhibit farnesyl transferase, a preparation method thereof, and a use as an anticancer agent.

In the present invention, there is provided a trans-pyrrolidine derivative and a compound of formula (I) having an anticancer effect by inhibiting panesyl transferase, and a pharmaceutically acceptable salt thereof.

[Formula 1]

Wherein R ₁ is selected from lower alkyl, aromatic, aromatic substituted lower alkyl, aromatic substituted halogen, dicyclic aromatic or aromatic containing nitrogen and sulfur atoms. R ₂ is selected from lower alkyl, aromatic, aromatic substituted by lower alkyl, aromatic substituted by halogen, aromatic or dicyclic aromatic containing nitrogen and sulfur atoms.

R ₃ may be represented by the following formula (2) or an amino acid.

[Formula 2]

In the general formula 2 A is a halogen, CN, NO _2, COOH, amide, thioamide, SR, and lower alkyl is substituted aromatic, halogen, CN, NO _2, COOH, amide, thioamide, SR and lower alkyl-substituted N and sulfur atoms are selected from the lower alkyl or the aromatic substituted in the ring, n is selected from 0 to 4.

As used herein, the term lower alkyl refers to straight or branched chain alkyl of 1 to 4 carbon atoms including methyl, ethyl, isopropyl, isobutyl, t-butyl.

As used herein, the term nitrogen and sulfur atom-containing aromatics are mono or dicyclic aromatic means that one to two nitrogen or sulfur atoms are contained in the aromatic ring.

The compounds of the present invention are those in which R ₁ and R ₂ are in a trans relationship with each other, have an asymmetric carbon center, may exist as racemates, racemic compounds, diastereomeric mixtures and individual diastereomers, all of these isomers Forms are included in the present invention.

Representative compounds of the present invention include the following substances.

1) 3- (trans-3,4-diphenylpyrrolidin-1-yl) -propionnitrile

2) 3- (trans-3,4-diphenylpyrrolidin-1-ylmethyl) -pyridine

3) 4- [4- (trans-3,4-diphenylpyrrolidin-1-yl) -3-imidazol-1-yl-butoxymethyl] -benzonitrile

4) 4- (trans-3,4-diphenylpyrrolidin-1-yl) -3-imidazol-1-yl-4-oxo-butyric acid ethyl ester

5) 4- [5- (trans-3,4-diphenylpyrrolidin-1-ylmethyl) -imidazol-1-yl-methyl] -benzonitrile

6) 4- {5- [2- (trans-3,4-diphenylpyrrolidin-1-yl) -ethyl] -imidazol-1-yl-methyl} -benzonitrile

7)

8) 4- [5- (trans-3,4-dinaphthalenepyrrolidin-1-ylmethyl) -imidazol-1-yl-methyl] -benzonitrile

9) 3- (trans-3-biphenyl-4-yl-4-phenylpyrrolidin-1-ylmethyl) -pyridine

In addition, the present invention provides a method for preparing a trans-pyrrolidine derivative.

The preparation method of the compound of the present invention can be summarized in the following steps.

1) substitution reaction of 2-bromoacetophenone to introduce a desired substituent (step 1),

2) cyclizing the compound prepared in step 1 (step 2),

3) obtaining a pyrrolidine ring compound by reducing the pyrrole ring compound prepared in step 2 (step 3),

4) reducing the compound prepared in step 3 (step 4),

5) (debenzylation) step (step 5) of removing the benzyl group of the compound prepared in step 4 and

6) obtaining the target compound by performing ① substitution, ② reductive amination or ③ amide coupling and reduction reaction of the compound prepared in step 5 (step 6)

More specifically, the compounds of the present invention can be prepared as shown in Scheme 1 to Scheme 3 below.

Scheme 1

Scheme 1 shows a process for obtaining a trans-pyrrolidine compound. 2-Bromoacetophenone of formula (I) undergoes a substitution reaction to form a compound of formula (II) or (III), and cyclizes it to 1-benzyl-3,4- which is a compound of formula (IV). Diphenyl-1,5-dihydro-pyrrole-2-one is obtained. The compound of formula (IV) having a pyrrole ring is reduced to obtain a compound of formula (V) having a pyrrolidine ring, which is then reduced to obtain a compound of formula (VI). This compound is dibenzylated to obtain a compound of formula (VII), wherein the compound of formula (VII) undergoes substitution (a), reductive amination (b), amide coupling, reduction reaction (c), etc. (IX), (X) and (XI) are obtained. The compounds of the formulas (IX), (X) and (XI) are the compounds of formula (I) which are the target compounds of the present invention.

Scheme 2

Scheme 2 describes a method for synthesizing the imidazole derivative used in the final substitution reaction of Scheme 1, wherein complex protecting group reaction and acetylation reaction, chlorination reaction, deprotection group reaction and halogenation from hydroxymethyl imidazole hydrocloid It shows what is obtained through the reaction. This will be described in detail by Preparation Example 1 to be described later.

Scheme 3

Scheme 3 describes a method for synthesizing the imidazole derivative used in the final amide coupling reduction Mitsunobu reaction of Scheme 1, and shows that it is obtained from diethyl fumarate through addition reaction and hydrolysis reaction. This will be described in detail by Production Example 2 to be described later.

Hereinafter, the preparation method of the novel compound of the present invention will be described in detail. However, the following examples are only for illustrating the present invention and the present invention is not limited by the examples. Prior to the Examples, the preparation method of the materials used to prepare the compounds of the present invention will be described.

Preparation Example 1 Preparation of 1-p-cyanobenzyl-5-chloromethyl imidazole

(Step 1) Preparation of 1-trityl-4-hydroxymethyl imidazole

3.99 g (29.6 mmol) of hydroxymethyl imidazole hydrochloride was dissolved in 30 mL of dimethylformamide and 10 mL of trimethylamine, followed by a solution of 9.35 g (33.5 mmol) of dimethylformamide (110 mL) of chloride of triphenylmethyl. Was added slowly. After 2 hours, 500 mL of ice water was added, and the resulting solid was obtained. This solid was recrystallized from dioxane to give 8.82 g (87% yield) of the title compound.

mp 227-229 ° C.

(Step 2) Preparation of (1-trityl-4-hydroxymethyl) imidazole acetate

After putting 5.00 g (14.7 mmol) of the compound prepared in (Step 1) of Preparation Example 1 in 100 mL of pyridine, 1.65 g (16.2 mmol) of acetic anhydride was added thereto, followed by stirring at room temperature for 24 hours. Pyridine was removed by distillation under reduced pressure, dissolved in 200 mL of ethyl acetate, and then washed with 100 mL of brine. The organic solvent was distilled off under reduced pressure and chromatographed with dichloromethane / methanol to give 5.22 g (13.7 mmol, 93% yield) of the title compound.

¹ H NMR (CDCl ₃ ) δ 2.01 (3H, s), 4.95 (2H, s), 6.88 (1H, s), 7.08 (5H, s), 7.27 (10H, s), 7.45 (1H, s).

(Step 3) Preparation of a brominated salt of [1-trityl-3- (4-cyanobenzyl) -4-hydroxymethyl] imidazole acetate

After dissolving 5.00 g (13.1 mmol) of the compound prepared in (Step 2) in 20 mL of dichloromethane, 2.82 g (14.4 mmol) of 4-cyanobenzylbromide was added thereto, and the mixture was stirred at room temperature for 60 hours. The organic solvent was removed by distillation under reduced pressure, and chromatographed with dichloromethane / methanol to give 5.31 g (9.17 mmol, 70% yield) of the title compound.

¹ H NMR (CDCl ₃ + CD ₃ OD) δ 1.95 (3H, s), 4.95 (2H, s), 5.45 (2H, s), 7.11-7.40 (18H, m), 7.65 (2H, d), 8.21 (1H, s).

(Step 4) Preparation of [1- (4-cyanobenzyl) -5-hydroxymethyl] imidazole acetate

9.10 g (15.7 mmol) of the compound prepared in (Step 3) was dissolved in 500 mL of dichloromethane, and then 6.06 mL (78.7 mmol) of trichloroacetic acid and 12.5 mL (78.7 mmol) of triethylsilane were slowly added at 0 ° C. Then stirred at room temperature for 1 hour. The organic solvent was distilled off under reduced pressure, the pH was adjusted to 10 with saturated potassium carbonate (K ₂ CO ₃ ) solution, and extracted with 300 mL of ethyl acetate. The organic solvent was distilled off under reduced pressure and the residue was chromatographed with ethyl acetate to give 3.60 g (14.1 mmol, 90% yield) of the title compound.

¹ H NMR (CDCl ₃ ) δ 1.90 (3H, s), 4.97 (2H, s), 5.25 (2H, s), 7.14 (2H, d), 7.21 (1H, d), 7.67 (1H, s), 7.75 (2 H, d).

(Step 5) Preparation of 1- (4-cyanobenzyl) -5-hydroxymethyl imidazole

4.20 g (16.5 mmol) of the compound prepared in (Step 4) was dissolved in 200 mL of methanol, and then 4.50 g (32.9 mmol) of potassium carbonate was added thereto, followed by stirring at room temperature for 20 minutes. The organic solvent was distilled off under reduced pressure, extracted with 300 mL of ethyl acetate, and chromatographed with dichloromethane / methanol to give 3.19 g (15.0 mmol, 91% yield) of the title compound.

¹ H NMR (CDCl ₃ + CD ₃ OD) δ 4.28 (2H, s), 5.18 (2H, s), 6.84 (1H, s), 7.12 (2H, d), 7.42 (1H, s), 7.55 (2H , d).

(Step 6) Preparation of 1- (4-cyanobenzyl) -5-chloromethyl imidazole

3.00 g (14.1 mmol) of the compound prepared in (Step 5) was dissolved in 40 mL of chloroform, and then 5.02 mL (70.5 mmol) of thionyl chloride was slowly added at 0 ° C., followed by stirring at room temperature for 2 hours. The organic solvent was distilled off under reduced pressure, dissolved in 50 mL of ethyl acetate, washed with a saturated solution of sodium bicarbonate (NaHCO ₃ ), and the organic solvent was distilled off under reduced pressure to obtain 2.91 g (12.5 mmol, 89% yield) of the title compound. . This compound was used in (step 6) of Example 1 which will be described later immediately without purification.

Preparation Example 2 Preparation of 3- (ethoxycarbonyl) -2-imidazol-1-ylsuccinic acid

(Step 1) Preparation of Diethyl-2-imidazol-1-ylsuccinate

3.61 g (51.5 mmol) of imidazole and 2.53 g (25.7 mmol) of diethylfumalate were dissolved in 50 mL of acetonitrile and the solution was boiled for 4 days. The solvent was removed by distillation under reduced pressure, and column chromatography was performed with methylene chloride / methanol to obtain 2.0 g (8.3 mmol, 32% yield) of the title compound.

¹ H NMR (CDCl ₃ ) δ 7.55 (1H, s), 7.04 (1H, s), 6.97 (1H, s), 5.19 (1H, dd), 4.12 (4H, q), 3.20 (1H, dd), 2.93 (1 H, doublet), 1.95 (6 H, t).

(Step 2) Preparation of 3- (ethoxycarbonyl) -2-imidazol-1-ylsuccinic acid

2.0 g (8.3 mmol) of the compound prepared in (Step 1) of Preparation Example 2 was dispersed in 20 mL of water and then boiled for 18 hours. The solid obtained by cooling to room temperature was filtered under reduced pressure and washed with diethyl ether to obtain 1.4 g (6.6 mmol, 80% yield) of the title compound.

¹ H NMR (DMSO-d ₆ ) δ 7.99 (1H, s), 7.36 (1H, s), 7.03 (1H, s), 5.36 (1H, dd), 3.50 (2H, q), 3.28 (1H, dd ), 3.16 (1H, doublet), 1.15 (3H, t).

Example 1 Preparation of 4- [5- (trans-3,4-diphenylpyrrolidinyl-1-methyl) -imidazol-1-ylmethyl] -benzonitrile

(Step 1) Preparation of N-benzyl-N- (2-oxo-2-phenyl-ethyl) -2-phenyl-acetamide

0.45 g (2.0 mmol) of 2-bromoacetophenone was added to 3 mL of dimethyl formamide solution containing 0.21 g (2.0 mmol) of benzylamine and 0.6 mL (4.3 mmol) of triethylamine. Stirred at ambient temperature. To this solution was added 0.41 g (3.0 mmol) of phenylacetic acid, 0.45 g (3.0 mmol) of N-hydroxy benzotriazole and 0.58 g (3.0 mmol) of 3-ethyl- (dimethylamino) -propyl carbodiimide. Stir for hours. After removing dimethyl formamide, 50 mL of ethyl acetate was added and washed with 1 N hydrochloric acid solution (3 x 20 mL), saturated potassium carbonate solution (3 x 20 mL) and saturated sodium chloride solution (2 x 20 mL). Purification by chromatography using ethyl acetate / hexanes gave 0.33 g (0.96 mmol, yield 48%) of the title compound.

¹ H NMR (CDCl ₃ ) δ 7.90 (2H, d), 7.80 (0.6H, d), 7.05-7.65 (16.9H, m), 4.77 (2H, s), 4.72 (0.6H, s), 4.67 ( 2H, s), 4.60 (0.6H, s), 3.90 (2H, s), 3.64 (0.6H, s).

(Step 2) Preparation of 1-benzyl-3,4-diphenyl-1,5-dihydro-pyrrol-2-one

0.33 g (0.96 mmol) of the compound prepared in (Step 1) of Example 1 was dissolved in 5 mL of acetonitrile, and then 0.082 mL of 1,8-diazabicyclo [5.4.0] unde-7-cene (DBU). (0.6 mmol) was added and boiled under nitrogen for 3 hours. After distilling the organic solvent under reduced pressure, 20 mL of ethyl acetate was added thereto and washed with 1 N hydrochloric acid solution (3 x 20 mL), saturated potassium carbonate solution (3 x 20 mL), and saturated sodium chloride solution (2 x 20 mL). Purification by chromatography using ethyl acetate / hexanes gave 0.24 g (0.74 mmol, yield 77%) of the title compound.

¹ H NMR (CDCl ₃ ) δ 7.23-7.46 (15H, m), 4.77 (2H, s), 4.18 (2H, s).

(Step 3) Preparation of 1-benzyl-trans-3,4-diphenyl-pyrrolidin-2-one

0.24 g (0.74 mmol) of the compound prepared in (Step 2) of Example 1 was dispersed in 5 mL of methanol, and 0.54 g (22.2 mmol) of magnesium was added at 0 ° C. and stirred at 30 min. Then it was stirred under nitrogen for 5 hours at room temperature. 20 mL of 1 N hydrochloric acid solution was added, followed by 40 mL of ethyl acetate. The organic layer was washed with saturated potassium carbonate solution (3 × 20 mL) and saturated sodium chloride solution (2 × 20 mL), and then purified by chromatography using ethyl acetate / hexane to give 0.17 g (0.53 mmol, 72% yield) of the title compound. Got it.

¹ H NMR (CDCl ₃ ) δ 7.10-7.70 (15H, m), 4.77 (1H, d), 4.60 (1H, d), 3.90 (1H, d), 3.70 (1H, dd), 3.62 (1H, m ), 3.44 (1 H, d).

(Step 4) Preparation of 1-benzyl-trans-3,4-diphenyl-pyrrolidine

0.04 g (1.06 mmol) of lithium aluminum hydride was added to 5 mL of tetrahydrofuran and then boiled under nitrogen for 30 minutes. To a boiling solution was added 0.17 g (0.53 mmol) of the compound prepared in (Step 3) of Example 1 in 5 mL of tetrahydrofuran and boiled for 2 hours. After adding a saturated potassium carbonate solution, the precipitated solid was removed, and the product was eluted from the aqueous layer using ethyl acetate. The organic solvent was distilled off under reduced pressure, and then purified by chromatography using ethyl acetate / hexane to give 0.16 g (0.50 mmol, 95% yield) of the title compound.

¹ H NMR (CDCl ₃ ) δ 7.42 (5H, s), 7.05-7.18 (6H, m), 6.90 (2H, s), 6.84 (2H, d), 4.12 (2H, dd), 3.84 (1H, m ), 3.72 (1H, dd), 3.6 (1H, dd), 3.50 (1H, dd), 3.38 (1H, dd), 2.78 (1H, m).

(Step 5) Preparation of trans-3,4-diphenyl-pyrrolidine

0.16 g (0.50 mmol) of the compound prepared in Example 1 (Step 4) and 0.5 mL (5%) of formic acid were added to 10 mL of methanol containing 0.008 g (5%) of palladium adsorbed activated carbon. Stir under gas for 3 days. After palladium adsorption activated carbon was removed by vacuum filtration using selite, the organic solvent was removed by distillation under reduced pressure. Ethyl acetate was added, the organic layer was washed with saturated potassium carbonate solution (3 x 20 mL) and saturated sodium chloride solution (2 x 20 mL), and then purified by chromatography with ethyl acetate / methanol to give 0.01 g (0.05 mmol, Yield 10%).

¹ H NMR (CDCl ₃ ) δ 7.05-7.45 (10H, m), 3.62 (2H, dd), 3.39 (2H, dd), 3.15 (2H, dd).

(Step 6) Preparation of 4- [5- (trans-3,4-diphenylpyrrolidinyl-1-methyl) -imidazol-1-ylmethyl] -benzonitrile

0.01 g (0.05 mmol) of the compound prepared in (Step 5) of Example 1 and 0.014 g (0.05 mmol) of the compound prepared in (Step 6) of Preparation Example 1 were dissolved in 5 mL of dimethylformamide, and 60% 0.004 g (0.1 mmol) of sodium hydride were added at 0 ° C. After 3 hours, dimethylformamide was removed by distillation under reduced pressure, dissolved in 10 mL of ethyl acetate, and washed with 10 mL of saturated sodium chloride solution. The organic solvent was removed by distillation under reduced pressure, and column chromatography was performed with ethyl acetate / methanol to obtain 0.014 g (0.035 mmol, yield 70%) of the title compound.

¹ H NMR (CDCl ₃ ) δ 7.62 (2H, dd), 7.53 (1H, s), 7.10-7.30 (12H, m), 7.01 (1H, s), 5.46 (2H, s), 3.54 (2H, s ), 3.28 (2H, dd), 3.05 (2H, dd), 2.72 (2H, dd).

FAB MS: 419 (M + 1)

Example 2 Preparation of 3- (trans-3,4-diphenylpyrrolidinyl-1-ylmethyl) -pyridine

0.12 g (0.54 mmol) of the compound prepared in (Step 5) of Example 1 and 0.051 mL (0.54 mmol) of 3-pyridinecarboxyaldehyde were dissolved in 10 mL of methanol, and then the pH of the solution was adjusted to 5 using acetic acid. Fit. Add 0.1 g (1.6 mmol) of sodium cyanoborohydride and boil for 10 hours. After methanol was removed by distillation under reduced pressure, 20 mL of ethyl acetate and 10 mL of saturated potassium carbonate solution were added to elute the product with an organic solvent. The title compound was purified by column chromatography using ethyl acetate / methanol to give 0.089 g (0.43 mmol, 80% yield) of the title compound.

¹ H NMR (CDCl ₃ ) δ 8.75 (1H, s), 8.50 (1H, d), 7.76 (1H, d), 7.12-7.31 (11H, m), 3.77 (1H, d), 3.67 (1H, d ), 3.39 (2H, dd), 3.16 (2H, dd), 2.85 (2H, dd).

FAB MS: 315 (M + 1)

Example 3 Preparation of 3- (trans-3,4-diphenylpyrrolidin-1-yl) -propionnitrile

The reaction was carried out in a similar manner to Example 1, to obtain the title compound.

¹ H NMR (300 MHz, CDCl ₃ ): δ 7.14-7.50 (10H, m), 3.42 (2H, m), 3.21 (2H, t), 2.96 (3H, m), 2.86 (1H, m), 2.60 (2H, t)

FAB MS: 277 (M + 1)

Example 4 Preparation of 4- [4- (trans-3,4-diphenylpyrrolidin-1-yl) -3-imidazol-1-yl-butoxymethyl] -benzonitrile

The reaction was carried out in a similar manner to Example 1, to obtain the title compound.

¹ H NMR (300 MHz, CDCl ₃ ): δ 7.55 (2H, d), 7.02-7.34 (14H, m), 6.97 (1H, s), 5.70 (2H, s), 3.75 (1H, m), 3.35 (2H, m), 3.10 (2H, m), 2.83 (2H, m), 2.30 (2H, m), 2.21 (2H, m), 2.04 (2H, m)

FAB MS: 477 (M + 1)

Example 5 Preparation of 4- (trans-3,4-diphenylpyrrolidin-1-yl) -3-imidazol-1-yl-4-oxo-butyric acid ethyl ester

The reaction was carried out in a similar manner to Example 1, to obtain the title compound.

¹ H NMR (300 MHz, CDCl ₃ ): δ 7.76 (1H, s), 7.12-7.48 (12H, m), 4.40 (1H, m), 4.10 (2H, q), 3.62 (2H, dd), 3.39 (2H, dd), 3.20 (1H, dd), 3.15 (2H, dd), 2.93 (1H, dd), 1.95 (6H, t)

FAB MS: 418 (M + 1)

Example 6 Preparation of 4- {5- [2- (trans-3,4-diphenylpyrrolidin-1-yl) -ethyl] -imidazol-1-yl-methyl} -benzonitrile

The reaction was carried out in a similar manner to Example 1, to obtain the title compound.

¹ H NMR (300 MHz, CDCl ₃ ): δ 7.60 (2H, d), 7.50 (1H, s), 7.12-7.31 (10H, m), 7.09 (2H, d), 6.99 (1H, s), 5.18 (2H, s), 3.35 (2H, m), 3.11 (2H, t), 2.83 (3H, m), 2.72 (1H, m), 2.64 (2H, m)

FAB MS: 433 (M + 1)

Example 7 Preparation of 4- [5- (trans-3-naphthalen-1-yl-4-phenylpyrrolidin-1-ylmethyl) -imidazol-1-yl-methyl] -benzonitrile

The reaction was carried out in a similar manner to Example 1, to obtain the title compound.

¹ H NMR (300 MHz, CDCl ₃ ): δ 6.7-7.9 (18H, m), 5.46 (2H, s), 3.54 (2H, s), 3.28 (2H, m), 3.05 (2H, m), 2.72 (2H, m)

FAB MS: 469 (M + 1)

Example 8 Preparation of 4- (trans-3,4-dinaphthalenepyrrolidin-1-yl) -3-imidazol-1-yl-4-oxo-butyric acid ethyl ester

The reaction was carried out in a similar manner to Example 1, to obtain the title compound.

¹ H NMR (300 MHz, CDCl ₃ ): δ 6.83-7.95 (20H, m), 5.50 (2H, s), 3.48 (2H, s), 3.20 (2H, dd), 2.90 (2H, dd), 2.81 (2H, dd)

FAB MS: 519 (M + 1)

Example 9 Preparation of 3- (trans-3-biphenyl-4-yl-4-phenylpyrrolidin-1-ylmethyl) -pyridine

The reaction was carried out in a similar manner to Example 1, to obtain the title compound.

¹ H NMR (300 MHz, CDCl ₃ ): δ 8.67 (1H, s), 8.52 (1H, d), 7.77 (1H, d), 7.16-7.62 (15H, m), 3.81 (1H, d), 3.72 (1H, d), 3.46 (2H, m), 3.21 (2H, dd), 2.90 (2H, dd)

FAB MS: 391 (M + 1)

Ras farnesyl transferase inhibitory assay

In this experiment, Ras farnesyl transferase prepared by genetic recombination technology was improved by improving the method of Pompiano [Pompliano et al., Biochemistry 31. 3800 (1992)], Ras substrate (Ras-CVLS) protein Was purified by the previously reported method (Chung et al., Bichimica et Biophysica Acta 1129, 278 (1992)).

The enzymatic reaction was carried out in 50 μl of 50 mM sodium hippie buffer containing 25 mmol potassium chloride, 25 mmol magnesium chloride, 10 mmol DTT and 50 μmol zinc chloride and 1.5 μmol of Ras substrate protein, 0.15 μmol of Tritium-panesyl pyrophosphate and 4.5 nmol of panesyl transferase were used.

In detail, the reaction was continued for 30 minutes at 37 ° C. after the addition of farnesyl transferase, and the reaction was stopped by adding 1 ml of ethanol solution containing 1 M hydrochloric acid, and the resulting precipitate was hopper harvester for filter binding. (Hopper #FH 225V) was adsorbed onto the GF / B filter, washed with ethanol, and the dried filter was performed by measuring radioactivity using an LKB beta counter. The enzyme titer assay was performed in the state of substrate unsaturation where the concentrations of Ras substrate protein and panesyl enzyme showed quantitative titers.The synthesized compounds were dissolved in dimethyl sulfoxide (DMSO) solvent and added within 5% of the total reaction solution. Inhibitory activity was evaluated. Enzyme inhibition capacity was expressed as a percentage of the amount of farnesyl introduced in the presence of the sample to the farnesyl introduced into the Ras substrate protein in the absence of the sample, and the concentration at which 50% of enzyme activity was inhibited was determined as the IC ₅₀ of each sample. . Geranylgeranyl transferase for evaluating the selective inhibitory capacity of the sample is described by Shaber et al., J. Biol chem. 265: 14701 (1990)] was modified and used from the cerebellum, and the geranyl geranyl pyrophosphate and Ras-CVIL substrate proteins, which are specific substrates of geranyl geranyl transferase under conditions similar to the panesyl transferase reaction. The experiment was performed using.

Inhibitory Effect Analysis of Intracellular Ras Farnesyl Transferase

In this experiment, a rat2 cell line expressing C-Harvey-Ras protein having a transgenic activity by mutation was used, and the experimental method was described by Decrue et al. [Declue J. E. et. al., Cancer Research 51: 712 (1991). The experimental method will be described in detail below.

The transformed Rat2 fibro blast cell line was dispensed with 3 × 10 ⁵ cells in a 60 mm cell culture dish, incubated for 48 hours in a 37 ° C. cell incubator, grown to a density of 50% or more, and then treated. In this case, dimethyl sulfoxide (DMSO) was used as the sample solvent, and 1% dimethyl sulfoxide concentration was used for both the control and test groups. Four hours after the sample was treated, methionine labeled with 150 μCi radioisotope [35S] per ml of medium was added and incubated for 20 hours, followed by washing with physiological saline. Contains 1 ml of cold lysis buffer (5 mmol magnesium chloride, 1 mmol DTT, 1% NP 40, 1 mmol EDTA, 1 mmol PMSF, 2 μmol lupetin, 2 μmol pepstatinA and 2 μmol antipain for cell lysis) The supernatant in which cells were lysed was obtained by high-speed centrifugation (12,000 g x 5 minutes) using 50 mM sodium hippie buffer solution. Y13-259, a monoclonal antibody that specifically binds to Ras protein and standardizes to obtain quantitative results in immunoprecipitation reaction by measuring radioisotope labeling amount of the supernatant [Furth, ME et. al., J. Virol 43: 294 (1982)] and reacted at 4 ° C. for 15 hours. In this solution, a protein A-agarose suspension bound to Goth-derived immunoglobulin-bound antibody was added and reacted at 4 ° C. for 1 hour, and then the immunization precipitate was removed in a buffer solution (50 mmol). 50 mM tris chloride buffer containing sodium chloride, 0.5% sodium dioxy cholate, 0.5% NP 40 and 0.1% SDS). An electrophoretic method was used for the analysis of the precipitate, which was boiled in the electrophoretic sample buffer and subjected to electrophoresis using 13.5% SDS polyacrylamide gel. After electrophoresis, the gel was fixed, dried, and then exposed to an X-ray film and developed for printing. From the experimental results, the inhibitory effect of intracellular Ras farnesyl transferase was determined by measuring the intensity of the non-bound bands of the farnesyl bound bands of the Ras protein, and the concentration of 50% of the farnesyl binding inhibitors was determined as CIC ₅₀ . .

Table 1 below summarizes the inhibitory efficacy of representative compounds.

Compound number IC ₅₀ (μM) CIC ₅₀ (μM) One 84 ND 2 70 ND 3 2.2 10 4 15 ND 5 0.023 1.0 6 0.015 1.5 7 0.010 0.6 8 0.014 1.0 9 40 ND ** ND: Not measured.

The compounds of the present invention are novel trans-pyrrolidine derivatives that inhibit the action of Ras protein by inhibiting the action of panesyl transferase, an enzyme that transfers the panesyl group of Ras protein.

The trans-pyrrolidine derivative of the present invention can be usefully used as an anticancer agent by having an excellent ability to inhibit farnesyl transferase.

Claims (5)

Trans-pyrrolidine derivatives and pharmaceutically acceptable salts, hydrates or solvates thereof, characterized by the following formula (1). [Formula 1] In Chemical Formula 1 1) R ₁ may be selected from hydrogen, halogen, aromatic, aromatic substituted by lower alkyl, aromatic containing nitrogen and sulfur atom, 2) R ₂ may be selected from aromatics, aromatics substituted with lower alkyl, aromatics containing a nitrogen and sulfur atom or dicyclic aromatics, 3) R ₃ may be represented by the following Chemical Formula 2. [Formula 2] In Formula 2, A is aromatic substituted with halogen, CN, NO ₂ , COOH, amide, thioamide, SR and lower alkyl, or substituted halogen, CN, NO ₂ , COOH amide, thioamide, SR and lower alkyl. The nitrogen and sulfur atoms may be selected from aromatics contained in the ring or lower alkyl substituted with such aromatics. n may be selected from 0 to 4. The compound of claim 1, wherein 3- (trans-3,4-diphenylpyrrolidin-1-yl) -propionnitrile, 3- (trans-3,4-diphenylpyrrolidin-1-ylmethyl) -pyridine, 4- [4- (trans-3,4-diphenylpyrrolidin-1-yl) -3-imidazol-1-yl-butoxymethyl] -benzonitrile, 4- (trans-3,4-diphenylpyrrolidin-1-yl) -3-imidazol-1-yl-4-oxo-butyric acid ethyl ester, 4- [5- (trans-3,4-diphenylpyrrolidin-1-ylmethyl) -imidazol-1-yl-methyl] -benzonitrile, 4- {5- [2- (trans-3,4-diphenylpyrrolidin-1-yl) -ethyl] -imidazol-1-yl-methyl} -benzonitrile, 4- [5- (trans-3,4-Dinaphthalenepyrrolidin-1-ylmethyl) -imidazol-1-yl-methyl] -benzonitrile or Trans-pyrrolidine derivatives characterized by 3- (trans-3-biphenyl-4-yl-4-phenylpyrrolidin-1-ylmethyl) -pyridine and pharmaceutically acceptable salts, hydrates thereof or Solvates. 1) substitution reaction of 2-bromoacephenone to introduce a desired substituent (step 1), 2) cyclizing the compound prepared in step 1 (step 2), 3) obtaining a pyrrolidine ring compound by reducing the pyrrole ring compound prepared in step 2 (step 3), 4) reducing the compound prepared in step 3 (step 4), 5) (debenzylation) step (step 5) of removing the benzyl group of the compound prepared in step 4 and 6) trans-blood of the present invention, comprising the step (6 steps) of the compound prepared in step 5 to obtain the target compound by performing ① substitution, ② reductive amination or ③ amide coupling and reduction reaction. Process for the preparation of a lollidine derivative. Use of the trans-pyrrolidine derivative of claim 1 as a farnesyl transferase inhibitor. Use of the trans-pyrrolidine derivative of claim 1 as an anticancer agent.