KR20080058708A - Pyrazolidine compound for controlling activity of z-dna bonding protein - Google Patents

Abstract

A novel pyrazolidine compound is provided to inhibit the biological function of a Z-DNA bonding protein by being specifically coupled to the Z-DNA bonding protein, thereby being used as an anti-viral agent. A pharmaceutical composition for controlling activity of a Z-DNA bonding protein comprises a therapeutically effective amount of a pyrazolidine compound represented by a formula(1) or a pharmaceutically acceptable salt and a pharmaceutical carrier or an excipient. In the formula(1), R1 is C6-20 aryl; R2 is C5-20 aryl or C5-20 heterocycle including O or N; each X and Y is independently C or N. A pharmaceutical composition for treating or preventing fox virus or vaccinia virus comprises the compound of the formula(1) or the pharmaceutically acceptable salt thereof and the pharmaceutically acceptable carrier or the excipient.

Description

PYRAZOLIDINE COMPOUND FOR CONTROLLING ACTIVITY OF Z-DNA BONDING PROTEIN

1 is a graph showing the results of Biacoa binding assay for the compound of Sample 1.

FIG. 2 is a graph showing the results of Biacoa binding assays for the compound of Sample 2.

3 is a graph showing the results of Biacoa binding assays for the compound of Sample 3.

Figure 4 is a photograph showing the results of gel transfer assay for the reaction mixture of hZa _ADAR1 and DNA in the presence of Sample 1.

The present invention relates to a compound useful for antiviral treatment and prevention and a pharmaceutical composition containing the same, and more particularly to a compound useful for antiviral treatment and prevention controlling the activity of Z-DNA binding proteins essential for pathogenicity such as poxvirus. It relates to a pharmaceutical composition containing.

Since the discovery of Z-DNA in the 1970s, the biological role of Z-DNA has been revealed little by little. Z-DNA is closely related to pathogenicity in poxvirus, and E3L protein, which binds to Z-DNA of Vaccinia virus, plays an important role in pathogenesis and has anti-apoptotic activity.

In addition, E3L protein was found to be pathogenic only when the Z-DNA domain and Z-DNA binding protein are paired. Therefore, it has been suggested that the degradation or loss of Z-DNA binding activity in E3L protein can be used as a target to block the onset of the virus because it has a critical effect on the survival of the virus.

Therefore, it is believed that a compound that inhibits the binding of Z-DNA binding proteins such as E3L protein to Z-DNA has the potential to be developed as an antiviral agent. However, no compound has been developed so far.

An object of the present invention is to provide a compound or pharmaceutical composition containing the same for controlling the activity of the Z-DNA binding protein by interfering with the binding to Z-DNA.

In order to achieve the above object, the present invention provides a pharmaceutical composition comprising a pyrazolidine compound of formula (1) or a pharmaceutically acceptable salt thereof. More specifically, there is provided a pharmaceutical composition for controlling the activity of a Z-DNA binding protein comprising a therapeutically effective amount of a pyrazolidine compound of formula (1) or a pharmaceutically acceptable salt thereof.

Hereinafter, the present invention will be described in more detail.

The Z-DNA binding domain (Z alpha) of a protein called human Adenosine Deaminase Acting on double-stranded RNA1 (ADAR1) is structurally very similar to the Z alpha domain of the E3L protein of Bacteria virus. Recently, through the study of the crystal structure of the Z alpha domain of human ADAR1, the three-dimensional structure of the Z alpha domain that binds to Z-DNA was clearly identified. The present invention is a pyrazolidine compound of formula (1) which binds to the Z alpha domain by performing a structure-based virtual search and biophysical analysis of the compound database using the X-ray crystal structure of the Z alpha domain present in ADAR1. Developed. The compounds described herein can inhibit the biological function of the Z-DNA binding protein by specifically binding to the Z-DNA binding protein and preventing the protein from binding to the Z-DNA.

Wherein R ₁ is C ₆ -C ₂₀ substituted or unsubstituted aryl,

R ₂ is C ₅ to C ₂₀ aryl or C ₅ to C ₂₀ heterocycle interrupted by O or N;

X and Y are independently carbon or nitrogen,

Aryl is unsubstituted or substituted with one or more halogen, C ₁ -C ₆ alkoxy or amide.

Particularly preferred compounds among the compounds of Formula 1 are shown in Table 1 below.

Sample Compound structure Compound name One

(2- (5-chloro-2-methoxybenzyl) pyrazolidin-1-yl) (2- (pyridin-2-yl) -2H-tetrazol-5-yl) methanone 2

N, N '-(4- (2- (5-methyl-2-phenyl-2H-1,2,3-triazole-4-carbonyl) pyrazolidine-1-carbonyl) -1,2- Phenylene) diacetamide 3

N, N '-(4- (2- (3-methyl-1-phenyl-1H-pyrazole-4-carbonyl) pyrazolidine-1-carbonyl) -1,2-phenylene) diacetamide

The present invention includes all of the pyrazolidine-based compounds represented by Formula 1 and pharmaceutically acceptable salts thereof, as well as possible solvates and hydrates that can be prepared therefrom.

The compound of formula 1 of the present invention may be used in the form of a pharmaceutically acceptable salt, and as the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. Organic acids and inorganic acids can be used as the free acid, hydrochloric acid, phosphoric acid, sulfuric acid and nitric acid can be used as the inorganic acid, and methanesulfonic acid, p-toluenesulfonic acid, acetic acid, trifluoroacetic acid, citric acid and maleic acid (maleic) can be used as the organic acid. acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid, propionic acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutamic acid Tartaric acid (glutaric acid), glucuronic acid (glucuronic acid), aspartic acid, ascorbic acid, carboxylic acid, vanillic acid, iodic acid and the like can be used.

The acid addition salts according to the invention can be prepared by conventional methods, for example by dissolving a compound of formula 1 in an excess aqueous solution of an acid and using a water miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. It can be prepared by precipitation. Equivalent amounts of the compound of formula (1) and acid or alcohol (eg, glycol monomethyl ether) in water may be heated and then the mixture is dried or the precipitated salt is filtered off with suction.

In addition, the compound of formula 1 is dissolved using a possible organic solvent such as diethyl ether, tetrahydrofuran, dichloromethane, acetonitrile, and the various inorganic and organic acids listed above are dissolved directly or in an organic solvent. It can also be prepared by precipitating the salt which is produced by addition in the form and suction suction.

The compound of formula 1 may be administered in various oral and parenteral dosage forms during clinical administration, and when formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc., which are commonly used, may be used. It is prepared. Solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, which solid preparations contain at least one excipient such as starch, calcium carbonate Sucrose (Sucrose) or lactose (Lactose), gelatin, etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Liquid preparations for oral administration include suspensions, solution solutions, emulsion syrups, and the like, 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, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerol, gelatin and the like can be used.

The dosage of the active ingredient according to the present invention is appropriately selected according to the absorption of the active ingredient in the body, the rate of water activation and excretion, the age, sex and condition of the patient, and the severity of the disease to be treated, but in general, 1 day The compound of formula 1 per kg of body weight can be administered once to several times in an amount of 0.1 to 500 mg, preferably 1 to 20 mg.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are illustrative of the present invention, and the content of the present invention is not limited by the examples.

Example 1 of the Compound of the Invention BIACOA Binding Assay

The compounds of Samples 1 to 3 described in Table 1 above were obtained commercially from Tripos Discovery Research Center, Bude-Stratton Business Park and Bude, Cornwall EX23 8LY UK.

hZa _ADAR1 (Zalpha domain present in human ADAR1 protein) was immobilized on the surface of the sensor chip and the system was stabilized by flowing a running buffer to the surface continuously. 40 mM of the compound of Samples 1 to 3 was injected to maintain the association phase for 2 minutes and then changed to experimental buffer to maintain the dissociation phase for 2 minutes. The surface of the sensor chip was then regenerated with 10 mM regeneration buffer and washed with 10 mM washing buffer. The experimental buffer was flowed before the next sample was injected. Experimental results are shown in FIGS. 1 to 3 and Table 2 below.

Sample number k _a (M ^-1 s ^-1 ) k _d (s ^-1 ) K _A (mM ^-1 ) K _D (mM) c ² One 8.55 10.4 x 10 ^-3 0.822 1.22 12 2 14.4 3.83 x10 ^-3 3.77 0.265 5.78 3 18.8 7.98 x10 ^-3 2.36 0.423 8.62

Example 2 Gel Migration Shift Assay

Oligonucleotide, d- (CG) ₁₂ , d-CA ₂ T ₃ (CG) ₂ A ₃ T ₂ G [γ ³² P] -ATP was labeled using polynucleotide kinase and buffer, and labeled oligonucleotides were purified by mini spin column and 20% PAGE. Binding of labeled oligonucleotides with hZa _ADAR1 was performed in DNA binding buffer. (15 mM Tris pH 7.9, 80 mM KCl, 4 mM DDT, 0.2 EDTA, 10% glycerol, 10 micrograms of bovine serum alumin) The reaction mixture was allowed to react at room temperature for 1 hour before being run on a 5% polyacrylamide gel. Youngphor was performed. After electrophoresis, the gel was transferred to a blotting paper and dried. The dried gel was exposed to a BAS image plate and radiographed with a BAS machine. The results are shown in FIG.

As described above, the compound of the present invention can inhibit the biological function of the Z-DNA binding protein by specifically binding to the Z-DNA binding protein and preventing the Z-DNA binding protein from binding to the Z-DNA. Because of this effect, the compounds of the present invention exhibit potential efficacy as antiviral agents.

Claims (6)

A pharmaceutical composition for controlling the activity of a Z-DNA binding protein comprising a therapeutically effective amount of a pyrazolidine compound of Formula 1, or a pharmaceutically acceptable salt thereof, together with a pharmaceutical carrier or excipient: Formula 1

Wherein R ₁ is C ₆ -C ₂₀ substituted or unsubstituted aryl, R ₂ is C ₅ to C ₂₀ aryl or C ₅ to C ₂₀ heterocycle which is interrupted by O or N; X and Y are independently carbon or nitrogen, Aryl is unsubstituted or substituted with one or more halogen, C ₁ -C ₆ alkoxy or amide. A pharmaceutical composition for antiviral treatment or prevention comprising a therapeutically effective amount of a pyrazolidine compound of Formula 1, or a pharmaceutically acceptable salt thereof, together with a pharmaceutical carrier or excipient. The pharmaceutical composition according to claim 2, wherein the virus is a pox virus or a vaccinia virus. The compound of formula (1) according to claim 1 or 2, wherein (2- (5-chloro-2-methoxybenzyl) pyrazolidin-1-yl) (2- (pyridin-2-yl) -2H-tetrazol-5-yl) methanone; N, N '-(4- (2- (5-methyl-2-phenyl-2H-1,2,3-triazole-4-carbonyl) pyrazolidine-1-carbonyl) -1,2- Phenylene) diacetamide; or N, N '-(4- (2- (3-methyl-1-phenyl-1H-pyrazole-4-carbonyl) pyrazolidine-1-carbonyl) -1,2-phenylene) diacetamide Phosphorus pharmaceutical composition. The pharmaceutical composition according to claim 1 or 2, which is a formulation for oral administration. The pharmaceutical composition according to claim 1 or 2, which is a formulation for parenteral administration.

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