KR20060116552A - N-formylhydroxylamine derivatives as matrix metalloproteinase inhibitors - Google Patents

Abstract

The present invention relates to a compound of formula (I) or a pharmaceutically acceptable salt useful as a matrix metalloproteinase (MMP) inhibitor, and a method for preparing the same, in particular, a gelatinase-based metalloproteinase ( N-pores, which selectively inhibit MMP-2 and MMP-9) and inhibit the invasion of pulmonary fibrous tumor cells (HT-1080) and tube formation of human umbilical vein endothelial cells (HUVECs). It relates to a method for producing a wheat hydroxylamine compound.

In Chemical Formula I,

R ₁ is hydrogen, straight or branched C _1-6 alkyl, halogen, hydroxy;

R ₂ is straight or branched C _1-6 alkyl, benzyl;

R ₃ is straight or branched C _1-6 alkyl, benzyl;

R ₄ is hydrogen, C _1-6 alkyl.

Description

N-formyl hydroxylamine derivatives as matrix metalloproteinase inhibitors}

1 is a result of analyzing the cytotoxicity of pulmonary fibrous tumor cells (HT-1080) by Example 4, Preparation Example 7 compound and the control material BB-94.

Figure 2 shows the results of analysis of the infiltration of lung fibroblast tumor cells (HT-1080) by Example 4, Preparation Example 7 compound and the control BB-94.

Figure 3 is a photograph analyzing the inhibition of angiogenesis of human umbilical vein endothelial cells (HUVEC) by Preparation Example 4, Preparation Example 7 compound and the control BB-94.

The present invention relates to novel N-formyl hydroxylamine derivatives that inhibit certain classes of matrix metalloproteinases (MMPs). More specifically, the present invention relates to novel N-formyl hydroxylamine derivatives or pharmaceutically acceptable salts as selective inhibitors of gelatinase-based metalloproteinases, and methods for their preparation.

Matrix metalloproteinases (hereinafter referred to as MMPs) are a group of enzymes that contain zinc ions (Zn ²⁺ ) at their active sites and play an important role in the degradation of extracellular matrix (ECM) components. to be. There are 23 known MMPs in humans, and the collagenase family, MMP-2 (gelatinase A) and MMP-9 (gelatinase B), belong to MMP-1 (collagenase-1) and MMP-8 (collagenase-2). Gelatinase family, MMP-3 (stromelysin-1) and MMP-10 (stromelysin-2) belong to the stromelysin family, MMP-14 (membrane type-1 MMP) belongs to the membrane-type MMP family . MMPs are a common process, such as embryonic development, wound repair and regeneration, where ECM degradation is necessary, as well as growth, invasion, metastasis, and angiogenesis of cancer cells by overexpression or excessive activation of MMPs. Important for pathological processes such as rheumatoid arthritis, osteoarthritis, abnormal osteoporosis, osteoporosis, periodontitis, interstitial nephritis, arteriosclerosis, emphysema, cirrhosis, corneal injury, autoimmune disease, or disease caused by invasion of white blood cells Known to play a role (Visse and Nagase, Circ.Res. 92: 827-839, 2003). Among the MMPs described above, gelatinase-based MMP-2 and MMP-9 decompose ECM components including type IV collagen, which is a main component of the basement membrane. It is known that gelatinase-based MMPs are important in these pathological processes, as basal membrane degradation is essential, particularly during cancer cell infiltration and metastasis and during neovascularization, which is required for cancer cell growth. Methods Enzymol. 248: 470-484, 1995; McCawley and Matrisian, Mol. Med. Today 6: 149-156, 2000).

Thus, MMP inhibitors are of importance in that they can be used as therapeutic agents for MMP related diseases as described above.

MMP inhibitors developed to date can be divided into three generations. The first generation of MMP inhibitors include batimastat (BB-94, British Biotech) and the like. Most of the MMPs have inhibitory effects but are not suitable for oral administration. Second-generation MMP inhibitors include marimastat (British Biotech) and prinomastat (Agouron Pharmaceuticals), which are not only inhibitory to most MMPs but can also be administered orally. These second-generation MMP inhibitors have a major disadvantage due to the characteristics that inhibit most MMPs, such as musculoskeletal pain during clinical application. In order to remedy this drawback, third generation MMP inhibitors that selectively inhibit some MMPs have been developed, and examples thereof include BAY12-9566 (Bayer Corporation).

The present inventors have tried to develop a novel substance having excellent selective inhibitory ability on MMPs of gelatinase family (MMP-2, MMP-9). As a result, the newly synthesized N-formyl hydroxylamine derivatives are MMP-2 and MMP- By selectively inhibiting the activity of 9 and inhibiting cancer cell invasion and angiogenesis of umbilical cord endothelial cells, the present invention was completed.

In the present invention, the following approach was used for the purpose of developing a novel substance having excellent selective inhibitory ability to MMP of gelatinase family (MMP-2, MMP-9).

First, to ensure selective inhibition of specific MMPs, N-formyl hydroxylamine, known as relatively weak ZBG, is used instead of the hydroxyxamic acid, a strong zinc binding group (ZBG) used in many MMP inhibitors. Was used. In addition, N-formyl hydroxylamine derivatives were synthesized through modification of the compounds R ₁ , R ₂ and R ₃ to bind to the S1, S1 'and S2' subsite pockets of the active site of MMP.

Second, recombinant MMP-1, MMP-2, MMP-9, MMP-3, and MMP-14 were prepared and the conditions for analysis of each MMP were established using fluorogenic oligopeptide as a substrate. The synthesized N-formyl hydroxylamine derivatives were analyzed for activity inhibition against recombinant MMP-1, MMP-2, MMP-9, MMP-3 and MMP-14. As a result, it was confirmed that the compound of Preparation Example 4 selectively inhibited MMP of gelatinase family (MMP-2 and MMP-9) and MMP-1 of collagenase family.

Third, when the R ₂ of the compound of Preparation Example 4 was modified from the above results, it was expected that the MMP of the gelatinase family could be more selectively inhibited. Thus, the new N-formyl hydroxylamine derivative was reflected in this result. Phosphorus Preparation Example 7 A compound was synthesized. As a result of analyzing the inhibitory activity against MMP-1, MMP-2, MMP-9, MMP-3, and MMP-14, the synthesized Example 7 compound was prepared in the gelatinase family. It was confirmed that MMP was selectively inhibited than the compound of Preparation Example 4.

Fourth, as a result of analyzing the effect on the cell growth of pulmonary fibrous tumor cells (HT-1080) to the compound of Preparation Example 4 and Preparation Example 7 to selectively inhibit gelatinase MMP, Preparation Example 4 And Preparation Example 7 It was confirmed that the compound is low in cytotoxicity compared to the reference drug BB-94. In addition, Preparation Example 4 and Preparation Example 7 compound, despite the selective inhibition of gelatinase-based MMP, invasiveness and renal blood vessels of pulmonary fibrous tumor cells (HT-1080) that can observe the cancer metastatic capacity In the tube formation analysis of umbilical vascular endothelial cells (HUVEC) whose production can be observed, the inhibitory effect is somewhat weaker than that of BB-94 which inhibits most MMPs (Compound 4) and comparable (Compound 7) Showed. Accordingly, the compounds of Preparation Example 4 and Preparation Example 7 which inhibit the selected gelatinase-based MMPs were confirmed to be MMP inhibitors that are highly applicable as therapeutic agents for MMP-related diseases including metastatic cancer.

After all, the main object of the present invention is to provide an N-formyl hydroxylamine derivative that selectively inhibits the gelatinase family of MMP activity. Another object of the present invention is to provide a method for producing an electrical derivative.

In Chemical Formula I,

R ₁ is hydrogen, straight or branched C _1-6 alkyl, halogen, hydroxy;

R ₂ is straight or branched C _1-6 alkyl, benzyl;

R ₃ is straight or branched C _1-6 alkyl, benzyl;

R ₄ is hydrogen, C _1-6 alkyl.

The compounds of the present invention may be in the form of racemic or optical isomers or diastereomers by containing the carbon free. Accordingly, the compounds of the present invention include all such racemates, optical isomers and diastereomers.

In addition, the compounds of the present invention may be in the form of pharmaceutically acceptable salts, hydrates, or solvates. Examples of pharmaceutically acceptable salts that can be applied to the compounds of the present invention include hydrochloride, bromate, sulfate, nitrate, methylsulfonate, p-toluenesulfonate, phosphate, acetate, pyruvate, citrate, succinate, lactate, Tartarate, fumarate, maleate, stearate, salicylate, sodium salt, potassium salt, magnesium salt, calcium salt and the like.

The present invention includes a process for preparing the compound of formula (I) or a pharmaceutically acceptable salt thereof.

That is, a method of preparing a compound of formula (I) and a pharmaceutically acceptable salt thereof comprising reacting a compound of formula (II) with a compound of formula (III) or a salt thereof followed by introducing a formyl group and removing a hydroxy protecting group do.

The reaction of the compound of formula II with the compound of formula III or a salt thereof may be carried out using conventional peptide synthesis methods such as penta-fluorophenol, N, O-dimethylhydroxylamine, DMAP-EDCI or EDCI-HOBt-. It can be carried out in the presence of a peptide binding reagent, such as NMM and available solvents include solvents such as tetrahydrofuran, dichloromethane, N, N- dimethylformamide. A protecting group such as benzyl can be carried out by adding a hydrogenation catalyst, preferably a palladium catalyst, to the amide product and stirring for about 2 to about 24 hours under a hydrogen atmosphere.

Wherein R ₁ , R ₂ , R ₃ and R ₄ are as defined above.

The present invention will be described with reference to the following examples, but these examples are for illustrative purposes only and should not be construed as limiting the scope of the invention.

Production Example

General Procedure: Synthesis of (R) -2-[(formyl-hydroxy-amino) -methyl] -4-methyl-pentanoic acid ((S) -alkyl) -amide (Scheme 1)

Step 1: (S) -4-benzyl-3- (4-methyl-pentanoyl) -oxazolidin-2-one (c)

Flask A: Compound a (R ₂ = iso-butyl, 20 g, 172.18 mmol) was dissolved in tetrahydrofuran (200 ml) dried under nitrogen atmosphere and cooled to -78 ° C to triethylamine (31.2 ml, 223.83 mmol) And trimethylacetyl chloride (23.3 ml, 189.4 mmol) were added, stirred for 30 minutes, stirred at room temperature for 2 hours, and cooled to -78 ° C. Flask B: Compound b ((S) -4-benzyl-oxazolidin-2-one, 36.6 g, 206.55 mmol) was dissolved in tetrahydrofuran (400 ml) dried under a nitrogen atmosphere, and cooled to -78 ° C. 2.5M n-butyllithium (82.65 ml, 206.55 mmol) was added and stirred for 1 hour. The reaction solution of Flask B was slowly added to the flask A reaction solution, followed by stirring at room temperature for 12 hours or more. The reaction mixture was treated with 1M KHCO ₃ (250 ml), concentrated under reduced pressure, diluted with ethyl acetate and washed with water. The organic layer was dried over magnesium sulfate, concentrated and purified by silica gel chromatography to obtain the title compound (42.6 g, 89.9%) in the form of an oil.

¹ H-NMR (CDCl ₃ ): δ 7.35-7.19 (m, 5H), 4.69-4.64 (m, 1H), 4.22-4.14 (m, 2H), 3.29 (dd, 1H), 2.98-2.90 (m, 2H), 2.80-2.72 (m, 1H), 1.67-1.54 (m, 3H), 0.94 (d, 6H).

Step 2: (S) -4-benzyl-3-((R) -2-benzyloxymethyl-4-methyl-pentanoyl) -oxazolidin-2-one (d)

Under nitrogen atmosphere, a solution of compound c (25 g, 90.79 mmol) in dried dichloromethane (350 ml) was cooled to 0 ° C. and 1M titanium chloride (99.9 ml, 99.87 mmol) was added. After 5 minutes, triethylamine (14.11 ml, 101.69 mmol) was added and stirred at 0 ° C. for 1 hour, benzyl chloromethyl ether (28.4 g, 181.6 mmol) was added, followed by stirring at 0 ° C. for 3 hours and at room temperature for 12 hours. The reaction mixture was treated with saturated NH ₄ Cl solution, the organic layer was separated, washed with water and saturated NaCl, dried over magnesium sulfate, concentrated and purified by silica gel chromatography to obtain the title compound (28.8 g, 80.2%) in oil form. .

¹ H-NMR (CDCl ₃ ): δ 7.32-7.16 (m, 10H), 4.73-4.68 (m, 1H), 4.54 (d, 2H), 4.38-4.36 (m, 1H), 4.19-4.08 (m, 2H), 3.77-3.62 (m, 2H), 3.22 (dd, 1H), 2.66-2.58 (m, 1H), 1.71-1.54 (m, 2H), 1.38-1.29 (m, 1H), 0.90 (dd, 6H).

Step 3: (R) -2-benzyloxymethyl-4-methyl-pentanoic acid (e)

In a nitrogen atmosphere, compound d (26.85 g, 67.89 mmol) was dissolved in a tetrahydrofuran / water (4/1, 420 ml) mixed solution, cooled to 0 ° C., 30% hydrogen peroxide (56 ml, 271.56 mmol) and lithium hydroxide. Seed (4.55 g, 110.22 mmol) was added and stirred for 3 hours. Treated with Na ₂ SO ₃ (34.3 g) solution dissolved in water (200 ml), concentrated under reduced pressure, washed with dichloromethane, and the aqueous layer was adjusted to PH = 2.5 with 2N aqueous hydrochloric acid solution. The aqueous layer was extracted with ethyl acetate, dried over magnesium sulfate and concentrated to give the title compound (15 g, 93.7%) in the form of an oil.

¹ H-NMR (CDCl ₃ ): δ 7.36-7.25 (m, 5H), 4.54 (s, 2H), 3.66-3.51 (m, 2H), 2.85-2.78 (m, 1H), 1.67-1.53 (m, 2H), 1.36-1.20 (m, 1H), 0.91 (dd, 6H).

Step 4: (R) -2-hydroxymethyl-4-methyl-pentanoic acid (f)

To a solution of compound e (14 g, 59.24 mmol) in ethyl alcohol (500 ml) was added 10% Pd / C (2 g) and stirred at room temperature for 20 hours. Filtration under celite and concentration gave the title compound (8.6 g, 99.3%) in oil form.

¹ H-NMR (CDCl ₃ ): δ 6.68 (s, 1.6H), 3.76 (d, 2H), 2.74-2.65 (m, 1H), 1.73-1.54 (m, 2H), 1.37-1.23 (m, 1H ), 0.93 (dd, 6H).

Step 5: (R) -2-hydroxymethyl-4-methyl-pentanoic acid benzyloxy amide (g)

Compound f (8 g, 54.72 mmol) was dissolved in dichloromethane (200 ml) and cooled to 0 ° C. to O-benzylhydroxylamine (9.17 g, 57.44 mmol), dimethylaminopyridine (DMAP, 13.37 g, 109.44 mmol), 1 -Ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI, 11.01 g, 57.43 mmol) was added and stirred at room temperature for 24 hours. 1N hydrochloric acid solution (220 ml) was added to the reaction mixture, extracted with dichloromethane, washed with water and saturated NaCl solution, dried over magnesium sulfate, and concentrated to give the title compound (11 g, 80%) as a white crystalline solid.

¹ H-NMR (CD ₃ OD): δ 7.44-7.32 (m, 5H), 4.86 (s, 2H), 3.66-3.43 (m, 2H), 3.31-3.29 (m, 1H), 2.23-2.24 (m , 1H), 1.52-1.38 (m, 2H), 1.18-1.11 (m, 1H) 0.85 (t, 6H).

Step 6: (R) -1-benzyloxy-3-isobutyl-azetidin-2-one (h)

In a nitrogen atmosphere, compound g (11 g, 43.77 mmol) was dissolved in dried tetrahydrofuran (400 ml) and cooled to 0 ° C. to triphenylphosphine (13.77 g, 52.5 mmol), diethylazodicarboxylate (DEAD , 9.15 g, 52.5 mmol) was added and stirred at room temperature for 18 hours. Water (400 ml) was added to the reaction mixture, extracted with ethyl acetate, washed with saturated NaCl solution, dried over magnesium sulfate, concentrated and purified by silica gel chromatography to obtain the title compound (8.7 g, 85.2%) in the form of an oil.

¹ H-NMR (CDCl ₃ ): δ 7.43-7.36 (m, 5H), 4.98 (s, 2H), 3.37 (t, 1H), 2.91-2.81 (m, 2H), 1.73-1.57 (m, 2H) , 1.34-1.23 (m, 1 H), 0.86 (dd, 6 H).

Step 7: (R) -2- (benzyloxyamino-methyl) -4-methyl-pentanoic acid (i)

To a solution of compound h (8 g, 34.29 mmol) in tetrahydrofuran / water / methyl alcohol (3/1/1, 400 ml) was added lithium hydroxide (14.39 g, 342.9 mmol) and stirred at room temperature for 18 hours. It was. Treated with water (200 ml), adjusted to pH = 2.5 with 3N hydrochloric acid solution, concentrated under reduced pressure, extracted with ethyl acetate, dried over magnesium sulfate and concentrated to give the title compound (8.5 g, 98.7%) in the form of an oil.

¹ H-NMR (CDCl ₃ ): δ 7.38-7.27 (m, 5H), 4.72 (d, 2H), 3.15-3.03 (m, 2H), 2.85-2.76 (m, 1H), 1.71-1.58 (m, 2H), 1.35-1.25 (m, 1H), 0.90 (t, 6H).

Step 8: (R) -2- (benzyloxyamino-methyl) -4-methyl-pentanoic acid ((S) -1-methylcarbamoyl-2-phenyl-ethyl) -amide (j)

Compound i (500 mg, 1.989 mmol) was dissolved in dimethylformamide (8 ml) and cooled to 0 ° C. to 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI, 457.7 mg, 2.387 mmol ), 1-hydroxybenzotriazole (HOBt, 322.6 mg, 2.387 mmol), N-methylmorpholine (NMM, 0.437 ml, 3.978 mmol) was added and stirred for 45 minutes. L-amino acid-alkylamide (R ₃ = benzyl, R ₄ = methyl, 1.5 eq) was added to the reaction solution, stirred at room temperature for 24 hours, diluted with ethyl acetate, 1N hydrochloric acid solution, saturated NaHCO ₃ , water, and saturated NaCl. After washing each with, dried over magnesium sulfate, concentrated and purified by silica gel chromatography to give the title compound (600 mg, 60.0%) as a white crystalline solid.

¹ H-NMR (CDCl ₃ ): δ 7.36-7.16 (m, 5H), 6.51 (d, 0.7H), 6.03 (s, 0.7H), 5.41 (s, 0.7H), 4.63-4.56 (m, 3H ), 3.12-2.78 (m, 4H), 2.7 (d, 3H), 2.57-2.48 (m, 1H), 1.57-1.44 (m, 2H), 1.13-1.09 (m, 1H), 0.84 (dd, 6H) ).

Step 9: (R) -2-[(benzyloxy-formyl-amino) -methyl] -4-methyl-pentanoic acid ((S) -1-methylcarbamoyl-2-phenyl-ethyl) -amide ( k)

The solution of compound j (100 mg, 0.265 mmol) in dichloromethane (2 ml) was cooled to 0 ° C., formic acid (1.3 ml) and acetic anhydride (0.26 ml, 2.81 mmol) were added, followed by stirring at 0 ° C. for 3 hours. Concentrated under reduced pressure. Diluted again with dichloromethane, washed with saturated NaCl, dried over magnesium sulfate, concentrated and purified by silica gel chromatography to obtain the title compound (96 mg, 89.7%) as a white crystalline solid.

¹ H-NMR (CDCl ₃ ): δ 7.99 (s, 0.8H), 7.36-7.08 (m, 10H), 6.26 (s, 1H), 5.85 (s, 0.7H), 4.72 (s, 1.4H), 4.51-4.49 (m, 1H), 3.82-3.61 (m, 2H), 2.99-2.54 (m, 2H), 2.67-2.60 (m, 4H), 1.55-1.35 (m, 2H), 1.25-1.15 (m , 1H), 0.86-0.83 (m, 6H).

Step 10: (R) -2-[(Formyl-hydroxy-amino) -methyl] -4-methyl-pentanoic acid ((S) -1-methylcarbamoyl-2-phenyl-ethyl) -amide ( l)

To a solution of compound k (60 mg, 0.148 mmol) in ethyl alcohol (3 ml) was added 10% Pd / C (10 mg) and stirred at room temperature for 4 hours. Filtration under celite, concentration and purification by silica gel chromatography gave the title compound (30 mg, 65%) as a white or pale pink crystalline solid.

¹ H-NMR (CD ₃ OD): δ 8.13 (s, 0.42H), 7.79 (s, 0.48H), 7.29-7.19 (m, 5H), 4.53-4.48 (m, 1H), 3.68-3.29 (m , 2H), 3.06-2.75 (m, 3H), 2.63 (d, 3H), 1.49-1.23 (m, 2H), 1.20-1.08 (m, 1H), 0.91-0.80 (m, 6H).

Preparation Example 1. (S) -2-[(R) -2-Benzyl-3- (formyl-hydroxy-amino) -propionylamino] -3,3 N-trimethyl-butyrylamide

The title compound was prepared from (R) -2-benzyl-3-benzyloxyamino-propionic acid and (S) -2-amino-3,3 N-trimethyl-butyamide hydrochloride according to the above general procedure.

¹ H-NMR (CDCl ₃ ): δ 8.41 (s, 0.4H), 7.83 (s, 0.6H), 7.29-7.19 (m, 5H), 6.65 (d, 0.4H), 6.55 (d, 0.6H) , 4.91-4.83 (m, 1H), 4.03-3.95 (m, 0.4H), 3.84-3.74 (m, 0.6H), 3.62-3.43 (m, 1H), 3.16 (s, 1H), 3.13 (s, 2H), 2.89-2.79 (m, 0.6H), 2.76-2.71 (m, 0.4), 1.69-1.34 (m, 1.8), 1.29-1.20 (m, 1.2H), 1.01-0.95 (m, 9H).

Preparation Example 2. (2R, 3S) -2-Benzyl-N-((S) -2,2-dimethyl-1-methylcarbamoyl-propyl) -3- (formyl-hydroxy-amino)- Butyramide

The title compound was prepared from (2R, 3S) -2-benzyl-3-benzyloxyamino-butyric acid and (S) -2-amino-3,3 N-trimethyl-butyamide hydrochloride according to the above general procedure.

¹ H-NMR (CDCl ₃ ): δ 8.44 (s, 0.4H), 7.81 (s, 0.6H), 7.27-7.19 (m, 5H), 6.59 (d, 0.4H), 6.49 (d, 0.6H) , 4.90-4.83 (m, 1H), 4.01-3.95 (m, 0.4H), 3.81-3.74 (m, 0.6H), 3.15 (m, 1H), 3.09 (s, 2H), 2.92-2.79 (m, 0.6H), 2.76-2.71 (m, 0.4), 1.70-1.34 (m, 1.8), 1.30-1.20 (m, 1.2H), 1.16-1.12 (m, 3H), 1.01-0.95 (m, 9H).

Preparation Example 3. (2R, 3S) -2-Benzyl-3- (formyl-hydroxy-amino) -pentanoic acid ((S) -2,2-dimethyl-1-methylcarbamoyl-propyl)- amides

The title compound was prepared from (2R, 3S) -2-benzyl-3-benzyloxyamino-pentanoic acid and (S) -2-amino-3,3 N-trimethyl-butyramide hydrochloride according to the above general procedure.

¹ H-NMR (CDCl ₃ ): δ 8.42 (s, 0.4H), 7.80 (s, 0.6H), 7.28-7.16 (m, 5H), 6.64 (d, 0.4H), 6.54 (d, 0.6H) , 4.89-4.83 (m, 1H), 4.02-3.95 (m, 0.4H), 3.83-3.74 (m, 0.6H), 3.18 (m, 1H), 3.12 (s, 2H), 2.89-2.79 (m, 0.6H), 2.75-2.71 (m, 0.4), 1.69-1.34 (m, 1.8), 1.28-1.20 (m, 1.2H), 1.16-1.12 (m, 2H), 1.01-0.95 (m, 12H).

Preparation Example 4. (R) -2-[(Formyl-hydroxy-amino) -methyl] -4-methyl-pentanoic acid ((S) -2,2-dimethyl-1-methylcarbamoyl-propyl )-amides

According to the above general procedure to prepare the title compound from (R) -2- (benzyloxyamino-methyl) -4-methyl-pentanoic acid and (S) -2-amino-3,3 N-trimethyl-butyamide hydrochloride It was.

¹ H-NMR (CDCl ₃ ): δ 8.47 (s, 0.4H), 7.89 (s, 0.6H), 6.66 (d, 0.4H), 6.51 (d, 0.6H), 4.90-4.85 (m, 1H) , 4.01-3.95 (m, 0.4H), 3.83-3.74 (m, 0.6H), 3.64-3.43 (m, 1H), 3.17 (s, 1H), 3.13 (s, 2H), 2.89-2.79 (m, 0.6H), 2.76-2.71 (m, 0.4), 1.69-1.34 (m, 1.8), 1.29-1.20 (m, 1.2H), 1.01-0.95 (m, 9H), 0.93-0.88 (m, 6H).

Preparation Example 5. (S) -2-[(R) -2-Benzyl-3- (formyl-hydroxy-amino) -propionylamino] -4-methyl-pentanoic acid methylamide

The title compound was prepared from (R) -2-benzyl-3-benzyloxyamino-propionic acid and (S) -2-amino-4-methyl-pentanoic acid methylamide hydrochloride according to the above general procedure.

¹ H-NMR (CDCl ₃ ): δ 8.39 (s, 0.4H), 7.81 (s, 0.6H), 7.28-7.18 (m, 5H), 6.63 (d, 0.4H), 6.45 (d, 0.6H) , 4.90-4.81 (m, 1H), 4.03-3.94 (m, 0.4H), 3.82-3.74 (m, 0.6H), 3.61-3.43 (m, 1H), 3.15 (s, 1H), 3.13 (s, 2H), 2.87-2.79 (m, 0.6H), 2.74-2.71 (m, 0.4), 1.67-1.34 (m, 1.8), 1.25-1.20 (m, 1.2H), 1.02-0.95 (m, 9H).

Preparation Example 6. (R) -2-Benzyl-3- (formyl-hydroxy-amino) -N-((S) -1-methylcarbamoyl-2-phenyl-ethyl) propionamide

The title compound was prepared from (R) -2-benzyl-3-benzyloxyamino-propionic acid and (S) -2-amino-N-methyl-3-phenyl-propionamide hydrochloride according to the above general procedure.

¹ H-NMR (CD ₃ OD): δ 8.15 (s, 0.42H), 7.82 (s, 0.48H), 7.31-7.18 (m, 10H), 4.55-4.47 (m, 1H), 3.67-3.31 (m , 2H), 3.04-2.75 (m, 3H), 2.62 (d, 3H), 1.48-1.25 (m, 1H), 1.20-1.08 (m, 1H).

Preparation Example 7. (R) -2-[(Formyl-hydroxy-amino) -methyl] -4-methyl-pentanoic acid ((S) -1-methylcarbamoyl-2-phenyl-ethyl)- amides

The title compound is prepared from (R) -2- (benzyloxyamino-methyl) -4-methyl-pentanoic acid and (S) -2-amino-N-methyl-3-phenyl-propionamide hydrochloride according to the above general procedure. It was.

¹ H-NMR (CD ₃ OD): δ 8.13 (s, 0.42H), 7.79 (s, 0.48H), 7.29-7.19 (m, 5H), 4.53-4.48 (m, 1H), 3.68-3.29 (m , 2H), 3.06-2.75 (m, 3H), 2.63 (d, 3H), 1.49-1.23 (m, 2H), 1.20-1.08 (m, 1H), 0.91-0.80 (m, 6H).

[Activity test]

Test Example 1 Preparation of MMP Enzyme

In order to obtain the MMP-14 catalytic domain, transcription initiation methionine (methionine), MMP-14 catalytic domains and the hinge domain (hinge domain, Tyr112-Ile318) of, and Escherichia coli (E a recombinant protein having six histidine marker (tag) coli BL21 (DE3)) in the form of inclusion bodies (Lee Seung-taek et al., Republic of Korea Application No. 10-2001-9899; Koo et al., Mol. Cells 13: 118-124, 2002). Inclusion bodies were dissolved in a buffer solution containing 6M urea and purified using a Ni ²⁺ -NTA resin affinity column (resin affinity column, Qiagen, Germany). The purified protein was refolded by dilution method, and 22.2 kDa MMP-14 catalytic domain having activity by cleavage of the hinge domain and histidine label by autocatalytic cleavage during the refolding process ( catalytic domain) (Lee Seung-taek et al., Korean Patent Registration No. 10-0456257; Koo et al., Mol. Cells 13: 118-124 (2002)).

In order to obtain the MMP-3 catalytic domain, a recombinant protein having a transcription initiation methionine and a catalytic domain of MMP-3 (Phe100-Pro273) was expressed in inclusion body in E. coli (see Ye et al., Biochemistry 31: 11231-11235 ( 1992). 21.8 kDa MMP- having active activity by refolding the inclusion body in a buffer solution containing 6 M urea and diluting it (Koo et al., Mol. Cells 13: 118-124, 2002). Three catalytic domains were obtained.

In order to obtain the MMP-1 catalytic domain, a recombinant protein having a transcription initiation methionine, a pro domain of MMP-1, a catalytic domain, a portion of the hinge domain (Gly261-Ala277), and a histidine label was expressed in inclusion body in E. coli. 30 kDa soluble MMP-1 pro, catalyst and refolded by diluting the inclusion body in a buffer solution containing 6 M urea (Koo et al., Mol. Cells 13: 118-124, 2002). Hin-domain (pro-catalytic-hinge domains) were obtained and converted to 20.5 kDa MMP-1 catalytic domain with activity by treatment with 1 mM APMA prior to use.

ProMMP-2 and proMMP-9 were expressed in insect cells (Sf9) using baculovirus system. Cell cultures containing proMMP-2 or proMMP-9 were purified by gelatin-Sepharose affinity column chromatography (Jo et al., J. Biochem. Mol. Biol. 32: 60-66, 1999). proMMP-2 and proMMP-9 were converted to MMP-2 and MMP-9 which had activity by treating 1 mM APMA before use.

Test Example 2: Analysis of MMP Inhibitory Activity of Compounds Using Fluorogenic Oligopeptide as Substrate

In order to analyze whether compounds inhibit MMP activity, fluorogenic oligopeptide cleavage assays were performed on MMP-1, MMP-2, MMP-3, MMP-9 and MMP-14 to determine the degree of catalytic activity of each MMP in the presence of the compound. (Yamamoto et al., J. Med. Chem. 41: 1209-1217, 1998). The catalytic activity of MMP-1, MMP-2 and MMP-14 was (7-methoxycoumarin-4-yl) -acetyl-Pro-Leu-Gly-Leu- (3- [2,4 dinitrophenyl] -L-2, 3 diaminopropionyl) -Ala-Arg-NH ₂ (1 mM; M1895, Bachem California Inc., USA) was used as a substrate, and the catalytic activity of MMP-3 and MMP-9 was (7-methoxycoumarin-4-yl) -acetyl-Arg-Pro-Tyr-Ala-Nva-Trp-Met-Lys- (3- [2,4-dinitro-phenyl] -L-2,3-diaminopropionyl) -NH ₂ (1 mM; M2105, Bachem California Inc., USA) was used as the substrate. 10 nM MMP, concentration-specific compound (10 μl dissolved in 100% DMSO) activated in MMP assay buffer (50 mM Tris / HCl, pH 7.5, 150 mM NaCl, 5 mM CaCl ₂ , 0.5 mM Zncl ₂ ) 1 μl of fluorogenic oligopeptide was added to prepare 100 μl of a reaction mixture, which was reacted at 37 ° C. for 30 minutes. After stopping the reaction by adding 100 μl of 0.2 M sodium acetate (pH 4.0) to the reaction mixture, the fluorescence emitted at 393 nm was measured by excitation at 328 nm using a fluorescence detector (Kontron SFM25 fluorometer, Italy). It was. In general, in order to analyze the inhibitory ability of each MMP, the compound was performed three times in duplicate at concentrations of 10 nM, 30 nM, 100 nM, 300 nM, 1 μM, and the like. As a control, BB-94, which is a hydroxyxamic acid derivative and inhibits most MMPs, was used.

Test Example 3: R in Compound _One , R ₂  And R ₃ MMP inhibitory effect

ZBG had N-formyl hydroxylamine and analyzed the MMP inhibitory effects of the compounds according to the types of R ₁ , R ₂ and R ₃ (Table 1). Comparison of each side chain is R ₁ is hydrogen (-H), R ₂ is benzyl (-CH ₂ C ₆ H ₅ ), R ₃ is t-butyl (-C (CH ₃ ) ₃ ) Preparation Example 1 Relative comparisons were made using the compound as a base compound.

First, on the basis of Preparation Example 1 having hydrogen at the R ₁ position, Preparation Example 2 having a small side chain (-CH ₃ ) and Preparation Example ₃ having a large side chain (-CH ₂ CH ₃ ) were compared. By doing so, we analyzed the MMP inhibition according to the size of the side chain. The effect of R ₁ was different depending on the type of MMP. However, in the case of gelatinase-based MMP-2 and MMP-9, as the size of R ₁ increased, the inhibitory ability decreased slightly.

Second, based on the compound of Preparation Example 1 having benzyl at the R ₂ position, the MMP inhibitory ability of Preparation Example 4 having t-butyl was analyzed. In all MMPs used for analysis, iso-butyl (-CH ₂ CH (CH ₃ ) ₂ ) showed better inhibition than benzyl (-CH ₂ C ₆ H ₅ ) and gelatinase-based MMP-2. MMP-9 was observed to be about 20 times better.

Third, Production Example 5 having iso-butyl (-CH ₂ CH (CH ₃ ) ₂ ) based on the compound of Preparation Example 1 having t-butyl (-C (CH ₃ ) ₃ ) at the R ₃ position The MMP inhibitory ability of the compound of Preparation Example 6 compound with the compound and benzyl (-CH ₂ C ₆ H ₅ ) was analyzed. The effect of R ₃ was different depending on the type of MMP. However, in the case of gelatinase-based MMP-2 and MMP-9, benzyl, t-butyl, and iso-butyl were shown to have excellent inhibition.

As a result of analyzing the compounds of Preparation Examples 1 to 6, which are N-formyl hydroxylamine derivative compounds, IC ₅₀ of the Preparation Example 4 compound for MMP-2 and MMP-9 was about 2 times higher than that of BB-94. was great, with respect to MMP-1 Preparation example 4 the compound was analyzed to be greater than the IC ₅₀ BB-94 to 10 times. However, Preparation Example 4 compound was very good relative selectivity to MMP-2, MMP-9 and MMP-1 compared to BB-94.

However, in the analysis results of the MMP inhibitory effect according to R ₁ , R ₂ and R ₃ in Table 1, MMP-2 and MMP-9, which are gelatinase-based MMPs, R ₁ is hydrogen, R ₂ iso-butyl, R ₃ was expected to be most efficiently inhibited in the presence of benzyl. As expected, for MMP-2 the IC ₅₀ (7.4 nM) of the compound of Preparation Example 7 decreased to less than 1/2 compared to the IC ₅₀ (18 nM) of the compound of Preparation Example 4, and for MMP-9 The IC ₅₀ (40 nM) of the compound of Preparation Example 7 slightly decreased compared to the IC ₅₀ (48 nM) of the compound of Preparation Example 4, but the IC ₅₀ (43 nM) of the compound of Preparation Example 7 for MMP-1 was prepared. Example 4 The compound's IC ₅₀ (25 nM) was nearly doubled. Therefore, the newly synthesized Preparation Example 7 compound increased the inhibitory activity against MMP-2 and MMP-9 and decreased the MMP-1 inhibition compared to the Preparation Example 4 compound. In addition, the IC ₅₀ (7.4 nM) of the compound of Preparation Example 7 for MMP-2 was at a level similar to the IC ₅₀ (8.9 nM) of BB-94, and the IC _{50 of the} compound of Preparation Example 7 for MMP-9 (40 nM) was less than twice the IC ₅₀ (24.5 nM) of BB-94, but the IC ₅₀ (43 nM) of the compound of Preparation Example 7 for MMP-1 compared to the IC ₅₀ (2.5 nM) of BB-94 It was 17 times more. Thus, the compound of Preparation Example 7 has a similar level of inhibition to gelatinase-based MMPs (MMP-2 and MMP-9) compared to BB-94, but also to MMP-1 as well as MMP-3 and MMP-14. Inhibition was very low. Based on these results, Preparation Example 7 compound was judged to have a very good selective inhibition of gelatinase-based MMP.

Table 1. MMP Inhibitory Effects of R ₁ , R ₂ , and R ₃ on Preparation Compounds

Test Example 4 Cytotoxicity Analysis by Preparation Example 4 and Preparation Example 7 Compound

In order to analyze the cytotoxicity of the compounds of Preparation Example 4 and Preparation Example 7, after treatment with the compound to the pulmonary fibrous tumor cells (HT-1080), the change in the number of cells was changed to 3- (4,5-dimethyl thiazol-2 -yl) -2,5-diphenyl tetrazolium bromide (MTT) analysis (Hansen et al., J. Immunol. Methods 119: 203 210, 1989). HT-1080 cells (10 ⁴ cell / well) were put in a 96 well dish and incubated in DMEM medium containing 10% FBS for 24 hours, and then Preparation Example 4 and Preparation Example 7 compounds and control BB-94 Was further incubated for 24 hours in DMEM medium containing concentrations (10, 30 and 100 μM) and 10% FBS. After incubation, 50 μl of 5 mg / ml MTT was treated in each well and incubated for 3 hours in a dark place. The solution was removed and the formazan produced by the living cells was lysed with 100% DMSO and the absorbance was measured at 565 nm. As can be seen from the results of the MTT assay, Preparation Example 4 and Preparation Example 7 compound was found to have a very small effect on the growth of HT-1080 cells, compared to BB-94, was less cytotoxic (Fig. 1).

Experimental Example 5: Invasion inhibition assay of pulmonary fibrous tumor cells by Preparation Example 4 and Preparation Example 7 compound

Invasion ability of cancer cells is essential in the process of cancer metastasis, and MMP-2 and MMP-9 are known to play an important role, especially in the process of basal membrane degradation (Murphy and Crabbe, Methods Enzymol. 248: 470 484, 1995). Therefore, in order to analyze the cancer metastasis inhibitory effect of the MMP inhibitor at the cellular level, the degree of invasion inhibition of pulmonary fibrous tumor cells (HT-1080) by Preparation Example 4, Preparation Example 7 compound and BB-94 by chemoinvasion measurement method Analyzed. 10 μl of 1 μg / μl gelatin was coated and dried on the bottom of the filter of Transwell (Corning, USA) with 8 μm pore and 85 μl of 0.35 μg / μl Matrigel (BD science, USA) was coated on the top of the filter and dried . HT-1080 cells grown subconfluent in DMEM medium containing 10% FBS were incubated with serum-free DMEM medium for 24 hours. The cells were separated by trypsin-EDTA, washed with serum-free DMEM medium, and suspended in 2.5 × 10 ⁵ cells / ml with the same medium. After dividing the cells by 200 μl, Preparation Example 4, Preparation Example 7 compound and BB-94 were added according to concentrations (final concentrations 0, 10, 30, and 100 μM), and reacted at room temperature for 1 hour. In the upper chamber, the suspension prepared by reacting Preparation Example 4, Preparation Example 7 compound and BB-94 was added. In the lower chamber, DMEM medium containing 10% FBS was added thereto, followed by incubation for 24 hours. Transwells were soaked in 100% methanol for 2 minutes to fix cells and stained with hematoxylin (Fisher Scientific, USA) and eosin-Y (Sigma, USA). Cells remaining on the top of the filter were removed with a cotton swab, and the number of infiltrated cells on the bottom was counted to analyze the effect of Preparation Example 4, Preparation Example 7 compound and BB-94 on cell infiltration.

As shown in Figure 2, Preparation Example 4, Preparation Example 7 compound and BB-94 inhibited the infiltration of HT-1080 cells in a concentration-dependent manner. Preparation Example 4 Compound was somewhat weaker than BB-94, Preparation Example 7 Compound was better than BB-94 and reduced the invasiveness of cancer cells, the concentration inhibiting 50% of invasiveness was 47.6 for Preparation Example 4 compound uM, Preparation Example 7 It was 17.8 uM for the compound and 33.4 uM for the BB-94. Therefore, Preparation Example 7 compounds as well as Preparation Example 4 compounds were expected to inhibit cancer cell infiltration through MMP inhibition at the cellular level.

Test Example 6 Analysis of Angiogenesis Inhibition of Umbilical Cord Blood Endothelial Cells (HUVEC) by Preparation Example 4 and Preparation Example 7 Compounds

New angiogenesis is known to be essential for processes such as cancer growth and metastasis (Jain, Science 307: 58-62, 2005). Umbilical cord vascular endothelial cells (HUVECs) form vascular structures in an MMP-dependent pattern when cultured on a basement-like Matrigel (Grant et al., Cell 58: 933-43, 1989; Wang and Keiser, Biochem. Biophys. Res. Commun. 272: 900-905, 2000). Therefore, the degree of inhibition of angiogenesis of umbilical cord endothelial cells (HUVEC) by Preparation Example 4, Preparation Example 7 compounds and BB-94 was analyzed. HUVEC was incubated in M199 medium (Gibco, USA) containing 20% FBS, 3 ng / ml basic FGF (Upstate, USA), 5 U / ml heparin (Sigma, USA) in a culture dish coated with 2% gelatin. . Subconfluent HUVECs were replaced with M199 medium containing 1% FBS, followed by depletion for 4 hours. The cells were separated by trypsin-EDTA, washed with M199 medium containing 1% FBS, and then suspended in 5 × 10 ⁵ cells / ml with the same medium. Add 250 μl of 10 mg / ml Matrigel (BD Biosciences, Bedford, USA) to each well of a 24 well dish and solidify, then add nothing to 5 X 10 ⁵ cell HUVEC in M199 medium containing 500 μl of 1% FBS. Or (negative control), 10 ng / ml VEGF (positive control), or 10 ng / ml VEGF and MMP inhibitor (BB-94, Preparation Example 4 or Preparation Example 7 compound by concentration) ) Was incubated for 18 hours. After cultivation was observed by taking a picture at 40 X magnification under a microscope.

As shown in FIG. 3, HUVECs cultured without additives on Matrigel showed a small amount of angiogenesis, whereas HUVECs treated with 10 ng / ml VEGF showed a thick vascular structure. It was observed that HUVEC treated with VEGF and Preparation Example 4, Preparation Example 7 compound or BB-94 at the same time inhibited angiogenesis in a concentration-dependent manner. In addition, it was confirmed that the effect of inhibiting angiogenesis was in the order of Preparation Example 7, BB-94, and Preparation Example 4 in the same order as the ability to inhibit gelatinase MMP. Therefore, the MMP inhibitor that selectively inhibits the gelatinase family of the present invention was expected to be excellent in inhibiting cancer metastasis through angiogenesis inhibition.

As described and demonstrated in detail as described above, the present invention selectively inhibits the MMP action of gelatinase family of MMP-2 (gelatinase A) and MMP-9 (gelatinase B), pulmonary fibrous tumor cells (HT N-formyl hydroxylamine derivative compounds, isomers thereof, and pharmaceutically acceptable salts and electric materials, which are excellent in inhibiting invasion and tube formation of umbilical vascular endothelial cells (HUVEC). to provide. The N-formyl hydroxylamine derivative compound of the present invention selectively inhibits the activity of MMP under in vitro conditions. Therefore, the MMP inhibitor comprising the N-formyl hydroxylamine derivative as an active ingredient is an MMP inhibitor. It may be useful for the prevention and treatment of various diseases caused by overexpression and excessive activation.

Claims (4)

Compound of Formula (I) or a pharmaceutically acceptable salt thereof:

In Chemical Formula I, R ₁ is hydrogen, straight or branched C _1-6 alkyl, halogen, hydroxy; R ₂ is straight or branched C _1-6 alkyl, benzyl; R ₃ is straight or branched C _1-6 alkyl, benzyl; R ₄ is hydrogen, C _1-6 alkyl. The compound of claim 1, wherein R ₁ is hydrogen, methyl, ethyl; R ₂ is n-butyl, iso-butyl, benzyl; R ₃ is iso-butyl, benzyl, t-butyl; R ₄ is methyl or a pharmaceutically acceptable salt thereof. A process for preparing a compound of formula (I) and a pharmaceutically acceptable salt thereof, comprising reacting a compound of formula (II) with a compound of formula (III) or a salt thereof followed by introducing a formyl group and removing a hydroxy protecting group.

Wherein R ₁ , R ₂ , R ₃ , and R ₄ are as defined in claim 1 above. The method according to claim 1, wherein (S) -2-[(R) -2-benzyl-3- (formyl-hydroxy-amino) -propionylamino] -3,3 N-trimethyl-butyramide; (2R, 3S) -2-Benzyl-N-((S) -2,2-dimethyl-1-methylcarbamoyl-propyl) -3- (formyl-hydroxy-amino) -butyramide; (2R, 3S) -2-Benzyl-3- (formyl-hydroxy-amino) -pentanoic acid ((S) -2,2-dimethyl-1-methylcarbamoyl-propyl) -amide; (R) -2-[(formyl-hydroxy-amino) -methyl] -4-methyl-pentanoic acid ((S) -2,2-dimethyl-1-methylcarbamoyl-propyl) -amide; (S) -2-[(R) -2-benzyl-3- (formyl-hydroxy-amino) -propionylamino] -4-methyl-pentanoic acid methylamide; (R) -2-benzyl-3- (formyl-hydroxy-amino) -N-((S) -1-methylcarbamoyl-2-phenyl-ethyl) propionamide; And From the group consisting of (R) -2-[(formyl-hydroxy-amino) -methyl] -4-methyl-pentanoic acid ((S) -1-methylcarbamoyl-2-phenyl-ethyl) -amide Selected compounds or pharmaceutically acceptable salts thereof.

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