KR20090058968A - A novel 4-(benzo[d][1,3]dioxol-5-ylamino)-4-oxobutanoic acid compound and the use as inhibitor of phenylethanolamin n-methyltransferase thereof - Google Patents

Abstract

A 4-(benzo[d][1,3]dioxol-5-ylamino)-4-oxobutanoic acid compound is provided to treat hypertension or depression by suppressing the generation of epinephrine. A 4-(benzo[d][1,3]dioxol-5-ylamino)-4-oxobutanoic acid compound is denoted by the chemical formula I. In the chemical formula I, R is -OH, -NH2, -SH, or -COOH. The compound is a compound inhibiting the phenylethanolamine N- methyltransferase. A pharmaceutical composition for preventing and treating hypertension comprises the compound as an active ingredient. The composition further comprises a pharmaceutically allowable carrier, diluents, or excipient.

Description

A novel 4- (benzo [d] [1,3] dioxol-5-ylamino) -4-oxobutanoic acid compound and its use as an inhibitor of phenylethanolamine N-methyl group transferase {A novel 4- (benzo [d] [1,3] dioxol-5-ylamino) -4-oxobutanoic acid compound and the use as inhibitor of Phenylethanolamin N-methyltransferase approximately}

The present invention provides 4- (benzo [d] [1,3] dioxol-5-ylamino) -4-oxobutanoic acid compound and its use as an inhibitor of phenylethanolamine N-methyltransferase and treatment of hypertension and depression. It is about applying to.

 In US Pat. No. 3,988,339, tetrahydroisoquinoline compounds have been found to be useful as inhibitors of phenylethanolamine N-methyl transferase. US Pat. No. 3,919,316 finds 2-aminodichlorotetraline useful as an inhibitor of phenylethanolamine N-methyltransferase. US Patent US4096173 has found that 1-amino-haloindan is useful as an antihypertensive agent by inhibiting phenylethanolamine N-methyl transferase. US Pat. No. 4,128,666 finds that 2-indanamine substituted with halogen is useful as an inhibitor of phenylethanolamine N-methyltransferase. In US Pat. No. 4,132737, 1-aminoindane substituted with trifluoromethyl group was found to be useful as an inhibitor of phenylethanolamine N-methyl transferase. Compounds of formula (I) found throughout the present invention are useful as inhibitors of phenylethanolamine N-methyltransferase and are novel.

An object of the present invention is to reduce the production of epinephrine by using a compound that inhibits phenylethanolamine N-methyl transferase, so that when epinephrine is excessively produced or the production of epinephrine is fatal, it is used for treating depression or hypertension.

In order to achieve the above object, the present invention provides a novel compound represented by the formula (I) and derivatives thereof.

<Formula I>

Wherein R is a hydrogen bond donor or a hydrogen bond acceptor, preferably R is -OH, -NH2, -SH, or -COOH, more preferably R is -COOH, or -OH, most preferably R is, but is not limited to, a novel 4- (benzo [d] [1,3] dioxol-5-ylamino) -4-oxobutanoic acid compound that is -COOH.

The present invention also provides a pharmaceutical composition for preventing and treating hypertension, comprising the compound of formula (I) as an active ingredient and a pharmaceutically acceptable carrier, diluent or excipient.

The present invention also provides a pharmaceutical composition for preventing and treating depression, comprising the compound of formula I as an active ingredient and comprising a pharmaceutically acceptable carrier, diluent or excipient.

The pharmaceutical composition for treating and preventing hypertension or depression of the present invention comprises the compound of Formula 1 as an active ingredient, according to a method easily known by those skilled in the art to which the invention pertains, It can be prepared in unit dosage form by formulating with an acceptable carrier, excipient or by incorporating into a multi-dose container. In this case, the formulation may be in the form of a solution or emulsion in an oil or an aqueous medium, or may be in the form of extracts, powders, granules, tablets or capsules, and may further include a dispersing or stabilizing agent.

The compound of formula 1 may be administered orally or parenterally during clinical administration and may be used in the form of a general pharmaceutical preparation.

That is, the compound represented by Formula 1 of the present invention can be administered in various oral and parenteral dosage forms during actual clinical administration, and when formulated, commonly used fillers, extenders, binders, wetting agents, disintegrants and surfactants It is prepared using diluents or excipients. Solid form preparations for oral administration include tablets, pills, powders, granules and capsules, and the like form at least one excipient such as starch, calcium carbonate, or the like. , Sucrose (Sucrose) or lactose (Lactose) and gelatin is mixed and prepared. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Oral liquid preparations include suspensions, liquid solutions, emulsions and syrups, and may include various excipients such as wetting agents, sweeteners, fragrances and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. .

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. As the non-aqueous solvent and suspending solvent, vegetable oils such as propylene glycol, polyethylene glycol and olive oil, injectable esters such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

According to the preparation method of the present invention, the method of administering the composition containing the purified compound is preferably oral or intravenous, and when the effective dose is oral, 50 to 500 mg per adult is usually used. Preferably, in the case of intravenous administration, 10 to 100 mg is preferred. Dosage levels for specific patients may vary depending on gender, age, health condition, diet, time of administration, method of administration, and drug severity and disease severity.

Hereinafter, the present invention will be described.

The present invention relates to novel uses of inhibitors that specifically inhibit phenylethanolamine N-methyltransferase in enzyme inhibitors. Phenylethanolamine N-methyltransferase is involved in the final step of biosynthesis of epinephrine (or adrenaline) by transferring methyl groups from S-adenosyl-L-methionine to norepinephrine (or noradrenaline). Epinephrine is a hormone synthesized in the adrenal medulla and released into the bloodstream in response to stress. Epinephrine makes you feel anxious, increases blood pressure and accelerates your heart rate. These changes can be fatal to patients with certain diseases such as angina pectoris, myocardial infarction and anxiety neuropathy. In addition, epinephrine is synthesized and released in the central nervous system to regulate blood pressure and heart rate. Compounds of formula 1 of the present invention (preferably 4- (benzo [d] [1,3] dioxol-5-ylamino) -4oxobutanoic acid) and their pharmaceutically acceptable compositions are phenylethanolamine N-methyl groups It inhibits the action of the transferase and ultimately reduces the production of epinephrine. Therefore, it is useful when the epinephrine is excessively produced or the production of epinephrine is fatal. These compounds are also useful for treating high blood pressure because they lower blood pressure and heart rate. The present invention relates to the use of compounds having the structure of formula (I) while inhibiting phenylethanolamine N-methyl transferase.

<Formula I>

Wherein R is a hydrogen bond donor or a hydrogen bond acceptor, preferably R is -OH, -NH2, -SH, or -COOH, more preferably R is -COOH, or -OH, most preferably R is, but is not limited to, a novel 4- (benzo [d] [1,3] dioxol-5-ylamino) -4-oxobutanoic acid compound that is -COOH.

 In the present invention, high performance liquid chromatography, nuclear magnetic resonance spectroscopy, and fluorescence spectrometry were used to evaluate the inhibitory ability of phenylethanolamine N-methyl transferase. The application of this technique to phenylethanolamine N-methyl transferase is novel. It belongs to the scope of the invention.

Inhibition ability evaluation techniques used in the present invention are liquid chromatography, nuclear magnetic resonance spectroscopy, fluorescence spectroscopy. Conventionally, ³ H-, ¹⁴ eoteuna to utilize a liquid scintillation using a SAM isotopically substituted in the C- and so on, which is very expensive and it is not easy to emit radiation generally used. Since the technique utilized in the present invention does not require substitution of such isotopes, it is inexpensive and has advantages in that anyone can use it.

As mentioned above, the present invention relates to the use of a compound that inhibits phenylethanolamine N-methyl transferase and is a novel invention. This ultimately reduces the production of epinephrine, which is useful when hyperephrine is excessively produced or when epinephrine production is fatal, and because it lowers blood pressure and heart rate.

Hereinafter, the present invention will be described in detail with reference to the following examples, which are presented to aid the understanding of the present invention, but the scope of the present invention is not limited to these examples in any sense.

Example 1 Expression of Phenylethanolamine N-Methyl Group Transferase

The cDNA of phenylethanolamine N-methyl transferase was donated by the 21st Century Frontier Human Gene Bank and inserted into the BamHI / XhoI restriction site of pET-28a (Novagen, Madison, Wis.), A Hexa-histidine-tagged expression vector. pET-28a-hPNMT-his vector was prepared and transformed with E. Coli strain BL21. E. Coli was incubated at 30 ° C. for 5 hours, dissolved in 20 mM sodium phosphate, 300 mM sodium chloride, pH 7.0 buffer (buffer A) and disrupted cells by sonication. Phenylethanol amine N-methyltransferase was isolated by centrifugation at 15,000 rpm for 20 minutes and buffer A containing 250 mM imidazole in a Ni-NTA column (Amersham Bioscience).

Example 2 Construction and Virtual Screening of 3D Compound Database

Using a list of compounds provided by Specs.net (Kluyverweg, Netherland), a three-dimensional structure database of 200,000 compounds was constructed.

Features such as hydrogen bond, hydrogen bond, and lipophilicity were used to determine Pharmacopore maps. Pharmacopore maps were generated by generating exclusion volumes of heavy atoms and determining the shape of the active site (Hoffren, AM et al. (2001), Curr. Pharm. Des. 7,547-566; Fisher, LS et al. (2002), J Braz. Chem. Soc. 13, 777-787; Kirchhoff, PD et al. (2001), J. Comp. Chem. 22, 993-1003.

Active sites were determined for the center and radius of bound inhibitors based on the X-ray structure of phenylethanolamine N-methyl transferase. An action model was generated within 10 Hz of the center of the active site. Among the multiple Pharmacopore maps, the compound database was searched by selecting the Pharmacopore map, which well describes the binding model of enzyme and inhibitor. All Pharmacopore maps were determined with three features consisting of one hydrogen bond acceptor, one hydrogen bond giver, and one lipophilic feature. Morphological constraints were used to control a large number of compounds (Fisher, LS et al. (2002), J. Braz. Chem. Soc. 13, 777-787; Kratov, EM et al. (2005) J. Chem. Inf. Model. 45, 146-159). This procedure used a Cerius ² / SBF module (Accelrys Inc., San Diego, Calif.). Inhibitor candidates were selected using visual inspection and LigScore (Venkatachalam, CM et al. (2003), J. Mol. Graph. Model. 21, 289-307; Rambabu, VAG, et al. (2005), J Chem. Inf.Model., 45, 725-738).

The results are shown in FIG.

Example 3 High Performance Liquid chromatography  Enzyme Inhibition Capacity Analysis

Norepinephrine and epinephrine were separated on a Waters Atlantis ^™ dC18 4.6 × 250 mm, 5 μm column. The mobile phase was H ₂ O: ACN: 100 mM ammonium acetate pH 5.0 = 10: 2: 88. The temperature of the column was kept at 25 ° C. and the UV detector was detected at 280 nm. To evaluate the enzyme inhibitory ability, 125 μL of 20 μM PNMT and 62.5 μL of 80 μM SAM were thoroughly mixed and aged at 37 ° C. for 1 hour. 5 μL of 50 mM inhibitor was added and aged at 37 ° C. for 15 minutes. 40 μL of norepinephrine 62.5 μL were added and aged at 37 ° C. for 15 minutes. The reaction was terminated by the addition of 20 μL perchloric acid, centrifuged at 6000 rpm for 5 minutes, and 10 μL of the supernatant was injected into high performance liquid chromatography. Using the area of the detected norepinephrine peak, the percentage of inhibition was calculated as follows.

Herein, the sample sample means a sample that is subjected to all the same processes as each sample but does not include an inhibitor. (I.e., norepinephrine is 100% biosynthesized with epinephrine)

As a result, it was confirmed that 4- (benzo [d] [1,3] dioxol-5-ylamino) -4-oxobutanoic acid inhibited 100% of phenylethanolamine N-methyltransferase. (Figure 1)

Example 4 Nuclear magnetic resonance spectroscopy  Enzyme Inhibition Capacity Analysis

Saturation Transfer Difference NMR (STD-NMR) experiments were performed at 298 K. The protein signal was saturated at -1.0 ppm and the total saturation time was 2 seconds. 10 μM Tris-Cl, 0.5 mM DTT, pH 7.0 buffer 10 μM of human PNMT and the inhibitor was dissolved to a concentration of 1: 100 and detected by removing the water signal using the WATERGATE sequence. As a result, it was confirmed that 4-oxo-1,4-dihydroquinoline-3,7-dicarboxylic acid inhibited phenylethanolamine N-methyl transferase 100%. (Figure 2)

Example 5 Fluorescence spectroscopy  Used Dissociation constant  Calculation

10 μM of human PNMT was dissolved in 10 mM Tris-Cl, 0.5 mM DTT, 1 mM EDTA pH 7.0 buffer and 20 μM SAM was added. While the inhibitor was added in small amounts, the concentration and fluorescence intensity were recorded as the inhibitor was added in small amounts until the final concentration of the protein and the inhibitor was 1: 100. The dissociation constant K _d was calculated using the following equation.

F0 and F are the intensity of the fluorescence signal detected at 324 nm with and without inhibitor, respectively, and n is the number of inhibitor binding surfaces in the protein.

The dissociation constant of 4- (benzo [d] [1,3] dioxol-5-ylamino) -4-oxobutanoic acid on PNMT was 3.24 × 10 ^-7 M. The experimental results are shown in FIG.

1 is a picture of the enzyme inhibition ability using a high performance liquid chromatography method, peak 1 in the figure is a control, peak 7 is 4- (benzo [d] [1,3] dioxol-5-ylamino) -4-oxobutanoic acid. As shown in the figure, 4- (benzo [d] [1,3] dioxol-5-ylamino) -4-oxobutanoic acid was found to inhibit 100% of phenylethanolamine N-methyltransferase.

FIG. 2 is a diagram analyzing enzyme inhibition ability using nuclear magnetic resonance spectroscopy. FIG. (A) shows STD-NMR of 4- (benzo [d] [1,3] dioxol-5-ylamino) -4-oxobutanoic acid. (B) shows the STD-NMR result of the non-binding sample. As can be seen from the figure, it was confirmed that 4-oxo-1,4-dihydroquinoline-3,7-dicarboxylic acid inhibited phenylethanolamine N-methyltransferase 100%.

3 is a diagram relating to the calculation of dissociation constants using fluorescence spectroscopy.

Figure 4 shows the screening results of the inhibitor of the present invention.

Claims (6)

New compounds represented by formula (I)

<Formula I> Wherein R is -OH, -NH ₂ , -SH, or -COOH. The compound of claim 1, wherein the compound is a 4- (benzo [d] [1,3] dioxol-5-ylamino) -4-oxobutanoic acid compound. The compound of claim 1 or 2, wherein the compound inhibits phenylethanolamine N-methyltransferase. The compound of claim 1 or 2, wherein the compound inhibits epinephrine production. A pharmaceutical composition for preventing and treating hypertension, comprising the compound of any one of claims 1 or 2 as an active ingredient and a pharmaceutically acceptable carrier, diluent or excipient. A pharmaceutical composition for preventing and treating depression, comprising the compound of any one of claims 1 or 2 as an active ingredient and a pharmaceutically acceptable carrier, diluent or excipient.

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