KR20030095893A - Pharmaceutical composition for inhibiting phosphoserine phosphatase activity comprising an amino-tetrahydro-benzo[b]thiopene-3-carboxylic acid derivative - Google Patents

Abstract

PURPOSE: Provided is a pharmaceutical composition comprising an amino-tetrahydro-benzo£b|thiopen-3-carboxylic acid derivative of the formula(1) for the inhibition of phosphoserine phosphatase(PSP) activity. Therefore, it is useful for the prevention and treatment of dementia and as a PSP inhibitor. CONSTITUTION: A pharmaceutical composition for the inhibition of phosphoserine phosphatase(PSP) activity is characterized by comprising an amino-tetrahydro-benzo£b|thiopen-3-carboxylic acid derivative of the formula(1), its pharmaceutically acceptable slat, hydrate, solvate or isomer, as an active ingredient. In the formula, X is CH2, O, S or NH; and R1 and R2 are individually hydrogen; linear or branched C1-C7 alkyl; or a hydroxy, halogen, alkyloxy, alkyl, amino, alkylamino, carboxylic acid or amide substituted or unsubstituted aromatic group.

Description

PHARMACEUTICAL COMPOSITION FOR INHIBITING PHOSPHOSERINE PHOSPHATASE ACTIVITY COMPRISING AN AMINO-TETRAHYDRO-BENZO [B] THIOPENE -3-CARBOXYLIC ACID DERIVATIVE}

The present invention relates to a pharmaceutical composition comprising a compound that inhibits the activity of phosphoserine phosphatase (PSP) as an active ingredient.

Human phosphoserine phosphatase is an enzyme that plays an important role in the biosynthetic pathway of L-serine. It is an enzyme that hydrolyzes phosphoserine to form serine and phosphoric acid. L-serine formed by phosphoserine is converted to D-serine by racemase in the brain, which is converted to glutamate as a neurotransmitter receptor in brain tissue, such as glutamate or glycerin. It acts as an agonist for N-methyl-D-aspartate (NMDA) receptors, a type of glutamate receptor.

PSP is known to be associated with Alzheimer's, a dementia (Heese et al, Neuroscience Letters 37: 288, 2000). Alzheimer's causes dementia by the formation of protein deposits known as beta amyloid in the brain, which in turn degenerate brain cells. It has been reported to inhibit (Heese et al, Neuroscience Letters 37: 288, 2000). Thus, PSP is believed to be associated with brain dysfunction in Alzheimer's by increasing cerebral toxicity by D-serine. The findings stem from the fact that the amount of serine in the brain tissue of patients with the disease is reduced.

Human phosphoserine phosphatase is composed of 225 amino acids and has 30% similarity to microorganism PSP such as E. coli, and it is known that magnesium is required for both the hydrolysis reaction of phosphoserine and phosphorylation of enzyme itself ( Collet et al., FEBS Letters 408: 281, 1997).

The inhibitors of PSP studied so far can be divided into two types, one is a phosphoserine mimetic, which binds to phosphoserine phosphatase competitively with phosphoserine and inhibits the activity of PSP, and the other is phosphoserine activity. Inhibits the activity of PSP by binding to a site other than the site (Hawkinson, European Journal of Pharmacology 337: 315, 1997).

2-amino-3-phosphonopropionic acid (AP3) has been reported as a compound that competitively binds to PSP with phosphoserine. As a compound to inhibit the L-alpha-glycerophosphocholine (La-glycerophosphorylcholine, GPC) and the like have been reported.

Accordingly, the present inventors have completed the present invention by selecting compounds capable of inhibiting the activity of PSP through the structural chemical genomics library, as a result of intensive studies to develop compounds that can prevent and treat dementia.

An object of the present invention is to provide a pharmaceutical composition comprising a compound that inhibits the activity of phosphoserine phosphatase as an active ingredient.

1 is a 2-amino-6- (1,1-dimethyl-propyl) -4,5,6,7-tetrahydro-benzo [b] thiophene-3-carboxylic acid amide compound and phosphoserine of the present invention. X-ray data analysis of the complex protein crystals of phosphatase are shown.

In order to achieve the above object, the present invention provides a composition for inhibiting phosphoserine phosphatase (PSP) comprising the compound of formula 1, a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof as an active ingredient to provide.

Formula 1

In which X is CH ₂ , O, S or NH,

R ¹ and R ² are each hydrogen; Straight or branched C ₁ -C ₇ alkyl; Or an aromatic group substituted or unsubstituted with hydroxy, halogen, alkyloxy, alkyl, amino, alkylamino, carboxylic acid or amide.

Compounds of formula (1) of the present invention can be obtained by building a structural chemical genomics library for phosphoserine phosphatase. The library is a technique based on structural chemical genomics that studies the process of recognizing chemicals that can be used for medical purposes based on three-dimensional structure. After collecting proteins with the protein group and grouping them into one protein group, compounds that can selectively bind to each protein group are selected by using virtual screening in the virtual space and collected in the form of a compound library. . Therefore, there is an advantage that by using the above method, when a protein having a corresponding compound library is already targeted, a leader can be secured at a very high speed.

Specifically, the first step is to collect all three-dimensional structures of phosphoserine phosphatase that can be obtained and classify them according to the structure of the active site. Mainly, three-dimensional structure information of a protein can be obtained from a protein data bank (PDB), or structural genomics studies can be obtained if necessary. The site where the new compound is in direct contact is then identified and the secondary structure of the new compound and the amino acids that may be involved in the contact are identified. In most cases, the active sites of the proteins belonging to the similar protein species contain almost the same parts, which means that the backbone of the compound as well as the side chain type and orientation of related residues are almost the same. In addition, each protein has a different structure, which can be used to increase the selectivity of new compounds.

The second step is the screening of chemical structures that recognize common structures of protein active sites on a computer using virtual screening ( in silico HTS). This technique is a technology to rapidly identify substances that can bind to the active site of the protein through docking experiments. The library obtained by performing the drug search method can be used to computerize the tertiary structure of the protein. After a high-speed binding test on the screen, the substances capable of binding to proteins belonging to the items of the protein active site structure database obtained therefrom are selected. In this process, amino-tetrahydro-benzo [b] thiophene-3-carboxylic acid (amino-tetrahydro-

It was confirmed that benzo [b] thiopene-3-carboxylic acid) can recognize the common structure of the active site of phosphoserine phosphatase.

The compound of formula 1 can then be obtained by collecting the compounds comprising amino-tetrahydro-benzo [b] thiophene-3-carboxylic acid as the last step.

Preferred among the compounds of formula (1) are those wherein X is N or O, R ¹ is C ₁ -C ₄ alkyl unsubstituted or substituted with C ₁ -C ₄ alkyl, and R ² is naphthalenyl or C ₁ -C ₄ alkyl Compound.

More preferred among the compounds of formula 1 of the present invention are the following compounds:

2-amino-6-methyl-4,5,6,7-tetrahydro-benzo [b] thiophene-3-carboxylic acid naphthalen-1-yl amide (2-Amino-6-methyl-4,5, 6,7-tetrahydro-benzo [b] thiophene-3-carbo

xylic acid naphthalen-1-yl amide)

2-Amino-6-tert-butyl-4,5,6,7-tetrahydro-benzo [b] thiophene-3-carboxylic acid ethyl ester (2-Amino-6-tert-butyl-4,5, 6,7-tetrahydro-benzo [b] thiophene-3-car

boxylic acid ethyl ester)

2-amino-6- (1,1-dimethyl-propyl) -4,5,6,7-tetrahydro-benzo [b] thiophene-3-carboxylic acid amide (2-Amino-6- (1, 1-dimethyl-propyl) -4,5,6,7-tetrahydro-benzo [b] thiophene-3-carboxylic acid amide)

Compounds of formula (I) of the present invention may have asymmetric carbon centers in their structure and therefore exist as individual enantiomers or diastereomers, and also as mixtures thereof, including racemates, such isomers or these Mixtures of are also included within the scope of the present invention.

The compounds according to the invention may also be provided in the form of pharmaceutically acceptable salts. Such pharmaceutically acceptable salts include alkali metal hydroxides (e.g. sodium hydroxide, potassium hydroxide), alkali metal bicarbonates (e.g. sodium bicarbonate, potassium bicarbonate), alkali metal carbonates (e.g. sodium carbonate, potassium carbonate, calcium carbonate), and the like. Inorganic bases and organic bases such as primary, secondary and tertiary amine amino acids are included.

The compounds of the invention may also be provided in the form of solvates, in particular hydrates. Hydration may occur during the separation of the compound, or may occur over time due to the hygroscopicity of the compound.

The compound of formula 1 of the present invention inhibits the activity of phosphoserine phosphatase, an enzyme associated with dementia, and exhibits an IC ₅₀ value of 200 μM or less.

Accordingly, the present invention provides a composition for the prevention and treatment of PSP inhibitors or dementia comprising the compound of formula 1, a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof as an active ingredient and a pharmaceutically acceptable carrier. to provide.

The compositions can be formulated in a variety of oral or parenteral dosage forms. Formulations for oral administration include, for example, tablets, capsules, and the like, which include, in addition to the active ingredient, diluents (e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine), glidants ( Eg silica, talc, stearic acid and its magnesium or calcium salts and / or polyethylene glycols). Tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine, optionally starch, agar, alginic acid or its Disintegrants or boiling mixtures such as sodium salts and / or absorbents, colorants, flavors and sweeteners. The formulations may be prepared by conventional mixing, granulating or coating methods. Also representative of parenteral formulations are injectable formulations, preferably aqueous isotonic solutions or suspensions.

The composition may contain sterile and / or auxiliaries such as preservatives, stabilizers, hydrating or emulsifying accelerators, salts and / or buffers for the control of osmotic pressure and other therapeutically useful substances and may be formulated according to conventional methods. have.

The pharmaceutical composition of the present invention may be parenterally or orally administered via a route such as intravenous, intramuscular, etc. as desired, and the compound of Formula 1 is preferably 0.01 to 100 mg / kg body weight per day, preferably A dose of 0.1 to 50 mg may be administered in one to several doses.The dosage level for a particular patient may be determined by the patient's weight, age, sex, health condition, diet, time of administration, method and excretion, and drug mixture and It can change depending on the severity of the disease.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Selection of PSP Inhibiting Compounds

(Step 1) PSP Active Site Structure Analysis

The first step in building a structural chemical genomics library for PSP is to collect all three-dimensional structures of available phosphatase and classify them according to the structure of the active site. To this end, all three-dimensional structural information of phosphoserine phosphatase is collected from a protein data bank (PDB), and the active site is collected using Acesly's Insight program on the Octane computer of SGI. Classified according to the structure of. The same computer and program were then used to analyze the amino acid residues and secondary structure of the active site, and to determine the site that would inhibit PSP activity when new compounds were in direct contact.

(Step 2) Selection of compounds that recognize the common structure of protein active sites

Next, after retrieving the chemical structure of recognizing the active site of PSP from MDL four ACD screening database were those with a drug discovery method (Virtual screening, in silico HTS) in a virtual space selected on the computer (noseonggu, Fine Chemicals 59:49, 2001). That is, a high-speed binding test was carried out on a computer for the tertiary structure of the protein in the virtual library of the virtual space obtained using the Aceli's Ludi program. In this process, amino-tetrahydro-benzo [b] thiophene-3-carboxylic acid in the structure of Formula 1

xylic acid) was able to recognize the common structure of the active site of phosphatase.

Finally, 2-amino-6-methyl-4, which is an amino-tetrahydro-benzo [b] thiophene-3-carboxylic acid as the active site identified above and which can bind to the active site of PSP, 5,6,7-tetrahydro-benzo [b] thiophene-3-carboxylic acid naphthalen-1-yl amide, 2-amino-6-tert-butyl-4,5,6,7-tetrahydro-benzo [b] thiophene-3-carboxylic acid ethyl ester and 2-amino-6- (1,1-dimethyl-propyl) -4,5,6,7-tetrahydro-benzo [b] thiophene-3- A carboxylic acid amide compound was obtained. The compounds are available from Specs and ChemDiv.

Example 2 PSP Activity Inhibitory Activity Analysis

The activity inhibitory activity of PSP was performed by modifying Cho et al. (PNAS 98: 8525, 2001). PSP used human PSP prepared as follows by gene recombination technology.

(2-1) Expression and Purification of PSP Proteins Including Histidine Labels

(Step 1) Amplification of PSP Gene

In order to synthesize and amplify the PSP gene including histidine tag through a polymerase chain reaction (PCR), primer oligonucleotides set forth in SEQ ID NOS: 1 and 2 were designed and synthesized using a nucleic acid synthesizer.

The primer of SEQ ID NO: 1 includes a nucleotide sequence corresponding to a BamHI restriction enzyme recognition site (GGATCC), and the primer of SEQ ID NO: 2 includes a nucleotide sequence corresponding to a stop codon (TTA) and a restriction enzyme XhoI recognition site (CTCGAG). do.

Human fetal liver cDNA library was used as a template in the same manner as in Example 1, and PCR was performed using the primer pairs of SEQ ID NOs: 1 and 2 as follows. The PCR reaction solution consisted of 1 μl human fetal liver cDNA library, 2 μl 10 mM dNTP (final concentration 0.2 mM), 3 μl 5 μM primers SEQ ID No. 1 and 2 (final concentration 0.3 μM), 0.5 μl EXT DNA polymerase (5 U / μl, DyNAzyme, USA) and 10 μl PCR buffer (DyNAzyme) were mixed and 80.5 μl of distilled water was added to adjust the final volume to 100 μl. The reaction solution was repeated for 15 minutes at 95 ° C, 1 minute 30 seconds at 94 ° C, 1 minute at 55 ° C, and 1 minute 30 seconds at 72 ° C. The reaction solution was separated by electrophoresis on a 1% agarose gel and eluted with about 500 bp of PSP gene. To 16 μl of the eluate, 2 μl of 10-fold restriction enzyme reaction buffer and 1 μl of restriction enzymes BamHI and XhoI were added and reacted at 37 ° C. for 2 hours. The reaction solution was then subjected to electrophoresis on a 1% agarose gel to extract the desired DNA fragment PSP B / X and then dissolved in 50 μl of distilled water.

(Step 2) Preparation of the expression vector containing the phosphoserine phosphatase gene

Plasmid pET21b (Novagen, USA) was digested completely with restriction enzymes BamHI and XhoI and separated on 1% agarose gel to recover plasmid pET21b B / X. 6 μg of the PSP B / X DNA fragment obtained in step 1 was added to the reaction tube along with 3 μg of plasmid pET21b B / X, followed by 2 μl of a 10-fold ligation reaction solution (500 mM Tris-HCl, pH 7.8, 100 mM magnesium chloride). , 100 mM DTT, 10 mM ATP) and 10 U of T4 DNA ligase were added and distilled water was added so that the total volume was 20 μl, and the reaction was carried out at 16 ° C. for 12 hours. The reaction solution was added to E. coli DH5α competent cells (Invitrogen, USA), transformed, and the E. coli transformants were selected by plating on 50 μg / ml LB-ampicillin medium. Plasmids were extracted from these, and restriction enzymes and sequencing confirmed that the PSP gene was inserted into the pET21b vector. The sequencing of the PSP gene cloned into the recombinant plasmid pET21b-PSP obtained above was carried out according to the method of Sanger et al. (Sanger, F. et al., PNAS USA 74: 5463, 1977). Big-Dye Cycle Sequencing System, Applied Biosystems, USA) and analyzed by ABI 377 DNA sequencer.

(Step 3) Expression of PSP in Escherichia Coli

The expression vector pET21b-PSP obtained in step 2 was transformed into E. coli BL21 (DE3) (Novagen Inc.), which is an expression host. The transformed E. coli strains were shaken for 12 hours in Luria medium (LB, 1% bactotryptone, 0.5% yeast extract, 1% sodium chloride) containing 100 μg / ml of ampicillin, and then 1 ml of 100 ml IPTG was added to a final concentration of 1 mM when the absorbance of the bacteria was transferred to Luria medium (containing 100 μg / ml of ampicillin) at 37 ° C. and about 0.5 at 600 nm. After 4 hours before and after IPTG addition, 1 ml each was taken and centrifuged at 10,000 g for 2 minutes to collect cell precipitates, respectively. The collected precipitate was suspended in PBS buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na ₂ HPO ₄ , 1.8 mM KH ₂ PO ₄ ), and the cells were crushed by an ultrasonic sonicator and then again at 10,000 g. Centrifuged for a minute. The separated supernatants and precipitates were electrophoresed on 15% SDS-polyacrylamide gels according to the method of Laemmli et al., Nature 227: 680, 1970, followed by protein extraction with Coomasie-Brilliant Blue. Staining was performed to analyze the expression level and sensitivity.

(Step 4) Isolation and Purification of PSP Protein from E. Coli Transformant

The PSP-expressing Escherichia coli transformants selected above were cultured in the same manner as in step 3, followed by centrifugation at 6,000 rpm for 10 minutes to collect cell precipitates, and a buffer solution of 50 mM Tris (pH 8.0) and 150 mM sodium chloride composition. After suspension in the ice bath cells were disrupted using an ultrasonic grinder (Sonic Dismembrator, Fisher, USA). The solution was centrifuged at 16,000 rpm for 30 minutes and the supernatant obtained therefrom was used for the following procedure.

The supernatant obtained above was bound to a nickel-nitrilotriacetic acid (Ni-NTA) column (Pharmacia, USA) previously equilibrated with the above buffer solution and eluted using a 0 to 0.5 M imidazole concentration gradient. After electrophoresis on the SDS-PAGE gel, only the PSP protein portion was collected and used in the following procedure. Concentrated 5 mL of the PSP protein obtained in the above process, and then gel filtration column (Superdex 75, 26/60) equilibrated with a buffer solution of 50 mM Tris (pH 7.5), 150 mM sodium chloride, 10% glycerol, 3 mM dTT composition , Pharmacia) and separated by the molecular weight of the protein was confirmed by electrophoresis and only the PSP protein portion was recovered.

(2-2) PSP activity inhibitory ability

2-Amino-6-methyl-4,5,6,7-tetrahydro-benzo [b] thiophene-3-carboxylic acid naphthalen-1-yl amide obtained in Example 1, 2-amino-6 Tert-butyl-4,5,6,7-tetrahydro-benzo [b] thiophene-3-carboxylic acid ethyl ester and 2-amino-6- (1,1-dimethyl-propyl) -4,5 Sample compounds were prepared by dissolving the, 6,7-tetrahydro-benzo [b] thiophene-3-carboxylic acid amide compound in dimethylsulfoxide (DMSO), respectively. Enzyme reactions were carried out in a buffer containing 25 mM Tris (pH 7.2) and 5 mM MgCl ₂ . First, 8 μl of the 1 mM phosphoserine solution prepared in Example (2-1) was added to a 98 well plate, and then 2 μl of each sample compound solution and 90 μl of 10 nM phosphoserine phosphatase solution dissolved in DMSO were added. Dripping into the wells. After reacting the well plate at 37 ° C. for 30 minutes, malachite: molybdate solution (10 ml distilled water, 2 ml of 1.3 mM malachite green dissolved in 3.6 M sulfuric acid, 0.5 ml 7.5% ammonium mol) 100 μl of ribdate and 40 μl 11% Tween 20) were added to the wells in reaction. After reacting the well plate for 50 minutes at room temperature, the absorbance at 630 nm was measured to evaluate the inhibitory ability of PSP activity of each sample compound. PSP activity inhibitory ability is a sample compound is free in solution in the free state was expressed as a percentage of the phosphate ion concentration measured in the presence sample compound for from phosphate ion concentration, the concentration of the compound to inhibit the enzymatic activity of _50% IC 50 values Was determined.

As a result, the compound 2-amino-6-methyl-4,5,6,7-tetrahydro-benzo [b] thiophene-3-carboxylic acid naphthalen-1-yl amide of the present invention, 2-amino-6 Tert-butyl-4,5,6,7-tetrahydro-benzo [b] thiophene-3-carboxylic acid ethyl ester and 2-amino-6- (1,1-dimethyl-propyl) -4,5 The, 6,7-tetrahydro-benzo [b] thiophene-3-carboxylic acid amides all had IC ₅₀ values of 200 μM or less.

Example 3 Binding Confirmation Using X-ray Crystallography

Compounds of the present invention identified in Example 2 that have the ability to inhibit PSP activity as a phosphoserine mimetic exhibits an inhibitory activity by binding to the PSP active site competitively with phosphoserine, or binds to a site other than the phosphoserine active site Human phosphoserine phosphatase used in Example 3 and 2-amino-6- (1,1-dimethyl-propyl) -4,5,6,7-tetrahydro- X-ray crystallography experiments were performed by combining the benzo [b] thiophene-3-carboxylic acid amides and making them into crystals. Protein crystallization was carried out using a drop mixing vapor equilibrium at 22 ° C., with phosphate (NaH ₂ HPO ₄ / K ₂ HPO ₄ ) as precipitant. High-speed nitrogen cooling was used to obtain high-resolution X-ray data, and the data were collected using radiation at the Pohang Accelerator Laboratory.

As a result, the 2-amino-6- (1,1-dimethyl-propyl) -4,5,6,7-tetrahydro-benzo [b] thiophene-3-carboxylic acid amide compound of the present invention is a human force. It was confirmed that PSP activity was inhibited by binding to the active site of porcelain phosphatase and acting as a phosphoserine mosafe. As shown in FIG. 1, when the complex structure was calculated using X-ray data of the complex protein crystal of the phosphoserine phosphatase and the inhibitor compound, it was confirmed that the compound binds to the active site of phosphoserine.

Since the compound of Formula 1 of the present invention effectively inhibits the activity of phosphoserine phosphatase, it may be usefully used as a pharmaceutical composition for the prevention and treatment of dementia.

Claims (4)

A composition for inhibiting phosphoserine phosphatase (PSP) containing a compound of Formula 1, a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof as an active ingredient. Formula 1 In which X is CH ₂ , O, S or NH, R ¹ and R ² are each hydrogen; Straight or branched C ₁ -C ₇ alkyl; Or an aromatic group substituted or unsubstituted with hydroxy, halogen, alkyloxy, alkyl, amino, alkylamino, carboxylic acid or amide. The method of claim 1, Containing a compound wherein X is N or O in Formula 1, R ¹ is C ₁ -C ₄ alkyl unsubstituted or substituted with C ₁ -C ₄ alkyl, and R ² is naphthalenyl or C ₁ -C ₄ alkyl A composition, characterized in that. The method of claim 1, A composition comprising a compound selected from the group consisting of: 2-amino-6-methyl-4,5,6,7-tetrahydro-benzo [b] thiophene-3-carboxylic acid naphthalen-1-yl amide, 2-amino-6-tert-butyl-4,5,6,7-tetrahydro-benzo [b] thiophene-3-carboxylic acid ethyl ester, and 2-Amino-6- (1,1-dimethyl-propyl) -4,5,6,7-tetrahydro-benzo [b] thiophene-3-carboxylic acid amide The method of claim 1, A composition, which is used for the prevention and treatment of dementia.