Phenylsulfonyl oxazole derivative and use thereof

Abstract

The present invention relates to a novel phenylsulfonyl oxazole derivative and a use thereof and specifically, to a compound represented by Chemical Formula 1 in the present specification or a pharmaceutically acceptable salt thereof, and to a use thereof for prevention, treatment, or improvement of neurodegenerative disease.

Claims

1. A compound represented by Chemical Formula 1 below or a pharmaceutically acceptable salt thereof: ##STR00010## wherein R is selected from the group consisting of halogen-substituted C1-C4 alkyl.

2. The compound or the pharmaceutically acceptable salt thereof of claim 1, wherein the R is trifluoromethyl (—CF.sub.3).

3. The compound or the pharmaceutically acceptable salt thereof of claim 1, wherein the compound is 5-methoxy-4-(phenylsulfonyl)-2-(4-(trifluoromethyl)phenyl)oxazole.

4. A composition comprising a compound represented by Chemical Formula 1 below or a pharmaceutically acceptable salt thereof as an active ingredient: ##STR00011## wherein R is selected from the group consisting of halogen-substituted C1-C4 alkyl.

5. The composition of claim 4, wherein the R is trifluoromethyl (—CF.sub.3).

6. The composition of claim 4, wherein the composition is a pharmaceutical composition or a food composition.

7. A method for treating neurodegenerative disease in a subject, comprising administering an effective amount of a composition comprising a compound represented by Chemical Formula 1 below or a pharmaceutically acceptable salt thereof as an active ingredient to the subject in need thereof: ##STR00012## wherein R is selected from the group consisting of halogen substituted C1-C4 alkyl.

8. The method of claim 7, wherein the neurodegenerative disease is at least one selected from the group consisting of Alzheimer's disease, Parkinson's disease, dementia, progressive supranuclear palsy, multi-system atrophy, olive-brain-cerebellar atrophy (OPCA), Shire-Dragger syndrome, striatonigral degeneration, Huntington's disease, amyotrophic lateral sclerosis (ALS), essential tremor, corticobasal degeneration, diffuse Lewy body disease, Parkin's-ALS-dementia complex, pick disease, cerebral ischemia, and cerebral infarction.

9. The method of claim 7, wherein the R is trifluoromethyl (—CF.sub.3).

10. The method of claim 7, wherein the compound is 5-methoxy-4-(phenylsulfonyl)-2-(4-(trifluoromethyl)phenyl)oxazole.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1 is a view illustrating an outline of an experiment performed to determine an effect of an autophagy enhancing compound on Alzheimer's disease.

(2) FIG. 2 illustrates a result of immunofluorescence staining of thioflavin S (ThioS, fibrillar amyloid beta plaques) in the brain cortex and the hippocampus of an APP/PS1 mouse injected with PBS or an autophagy enhancing compound and a result of quantifying areas occupied with the fibrillar amyloid beta plaques (n=4/group) (APP/PS1: Alzheimer's animal model).

(3) FIGS. 3A and 3B illustrate results of immunofluorescence staining and quantification of accumulation of Aβ40 (FIG. 3A) or Aβ42 (FIG. 3B) in the brain cortex and the hippocampus of an APP/PS1 mouse injected with PBS or an autophagy enhancing compound (n=4/group) (APP/PS1: Alzheimer's animal model).

(4) FIG. 4 illustrates results of immunofluorescence staining and quantification of accumulation of tau protein (AT8) in the brain cortex and the hippocampus of an APP/PS1 mouse injected with PBS or an autophagy enhancing compound (n=4/group) (APP/PS1: Alzheimer's animal model)

(5) FIGS. 5A to 5D illustrate results showing that the injection of an autophagy enhancing compound in an APP/PS1 mouse restores learning and cognitive functions (WT: wild type, APP/PS1: Alzheimer's animal model).

(6) FIG. 5A illustrates results of evaluating learning and memory through a Morris water maze test in a wild type mouse (n=6), an APP/PS1 mouse injected with PBS (n=6), and an APP/PS1 mouse injected with an autophagy enhancing compound (n=10).

(7) FIG. 5B illustrates a result showing a time left in a target platform on day 11 of the test.

(8) FIG. 5C illustrates the number of times of entering into a target area of the target platform on day 11 of the test.

(9) FIG. 5D illustrates results of contextual and tone tasks during a fear conditioning test.

(10) FIGS. 6A to 6C illustrate results of confirming that the increased neuroinflammation in an APP/PS1 mouse is reduced by the injection of the autophagy enhancing compound (n=3/group).

(11) FIG. 6A illustrates a result of quantifying areas of astrocytes (GFAP) through immunofluorescence staining in the brain cortices and the hippocampi of a wild type mouse (WT), an APP/PS1 mouse injected with PBS, and an APP/PS1 mouse injected with an autophagy enhancing compound.

(12) FIG. 6B illustrates a result of quantifying areas of microglia (Iba-1) through immunofluorescence staining in the brain cortices and the hippocampi of a wild type mouse (WT), an APP/PS1 mouse injected with PBS, and an APP/PS1 mouse injected with an autophagy enhancing compound.

(13) FIG. 6C illustrates a result of evaluating mRNA expression levels of inflammatory markers TNF-α, IL-1β, and IL-6 in the brain cortices and the hippocampi of a wild type mouse (WT), an APP/PS1 mouse injected with PBS, and an APP/PS1 mouse injected with an autophagy enhancing compound.

(14) FIG. 7 illustrates a result of graphing the expression of an autophagy-related protein which is analyzed using Western blotting and quantified in the brain cortices of a wild type mouse (WT), an APP/PS1 mouse injected with PBS, and an APP/PS1 mouse injected with an autophagy enhancing compound (n=3/group).

MODE FOR INVENTION

(15) Hereinafter, the present invention will be described in detail.

(16) However, the following Examples are just illustrative of the present invention, and the contents of the present invention are not limited to the following Examples.

Example 1: Preparation of Compounds

(17) Compounds corresponding to Chemical Formula 1 of the present invention were obtained by the following methods.

1-1. Preparation of 5-methoxy-4-(phenylsulfonyl)-2-(4-(trifluoromethyl)phenyl)oxazole

(18) 2-(2-chlorophenyl)-5-methoxy-4-(phenylsulfonyl)oxazole was prepared by the following steps (1) to (3).

(19) (1) Step 1: Preparation of methyl 2-(phenylsulfonyl)acetate

(20) ##STR00007##

(21) A solution of methyl bromoacetate (10 g, 65.38 mmol), benzenesulfinic acid and sodium salt (12.9 g, 78.4 mmol) mixed in ethanol (200 mL) was refluxed for 24 hours. Next, the excess solvent was removed under reduced pressure. Next, the reaction mixture was dissolved in dichloromethane (400 mL) and washed with water (2×200 mL) and brine (salt water, 50 mL). Next, an organic layer was dried in anhydrous Na.sub.2SO.sub.4 and concentrated under reduced pressure to obtain methyl 2-(phenylsulfonyl)acetate (13.5 g, 96%). The obtained compound was used for synthesis of a compound in a next step without a separate purification process.

(22) (2) Step 2: Preparation of methyl 2-diazo-2-(phenylsulfonyl)acetate

(23) ##STR00008##

(24) Triethylamine (7.0 g, 69.3 mmol) was added in a solution stirred with methyl 2-(phenylsulfonyl)acetate (13.5 g, 67.7 mmol) and 4-acetamidobenzenesulfonyl azide (16.65 g, 69.31 mmol) in acetonitrile (500 mL) at 0° C. Thereafter, the reaction mixture was stirred at room temperature for 24 hours, the reaction mixture was concentrated under reduced pressure, and the produced precipitate was stirred in a solution of ethyl acetate and n-hexane diluted at 1:1 (3×600 mL, 1:1 ethyl acetate:n-hexane), and then the mixed organic material was concentrated under reduced pressure. Thereafter, the mixture was purified by a column chromatography using ethyl acetate and n-hexane to obtain 15 g of a methyl 2-diazo-2-(phenylsulfonyl)acetate compound as a lemon yellow solid (99%).

(25) (3) Step 3: Preparation of 5-methoxy-4-(phenylsulfonyl)-2-(4-(trifluoromethyl)phenyl) oxazole

(26) ##STR00009##

(27) Methyl 2-diazo-2-(phenylsulfonyl)acetate (1.85 g, 7.71 mmol) was added to a solution in which 4-(trifluoromethyl)benzonitrile (1.2 g, 7.013 mmol) and rhodium (II) acetate (61.99 mg, 0.14 mmol) were refluxed in chloroform (20 ml). After addition, the reaction mixture was refluxed for 3 hours. The reaction mixture was concentrated under reduced pressure to obtain 1.5 g of 5-methoxy-4-(phenylsulfonyl)-2-(4-(trifluoromethyl)phenyl)oxazole as a white solid using a column chromatography (56%).

(28) 1H NMR (300 MHz, DMSO-d6): δ 8.06 (d, J=8.28 Hz, 2H), 7.95 (d, J=7.18 Hz, 2H), 7.88 (d, J=8.28 Hz, 2H), 7.76-7.61 (m, 3H), 4.30 (s, 3H).

Example 2: Confirmation of Effects for Prevention and Treatment on Neurodegenerative Disease

Experiment Method

1. Mouse and Experiment Outline

(29) A mouse experiment was approved by the Kyungpook National University Institutional Animal Care and Use Committee (IACUC). As an animal model of neurodegenerative disease (particularly, Alzheimer's disease), a transgenic mouse line overexpressing APPswe (hAPP695swe) and PS1 (presenilin-1M146V) based on a C57BL/6 mouse (Charles River, UK) was used. [hereinafter, APP/PS1 mouse (denoted by AD), GlaxoSmithKline Co., Ltd.]

(30) To confirm a therapeutic effect of the autophagy enhancing compound obtained in <Example 1>, an experimental substance was administered to the animal model according to the experimental outline (schedule) illustrated in FIG. 1. Specifically, PBS or the autophagy enhancing compound was intraperitoneally injected into a 6.5-month-old mouse 3 times a week at a dose of 50 mg/kg. After two months of the injection of the autophagy enhancing compound, behavioral analysis was performed, and the brain tissue of the mouse was used as a sample after the behavioral analysis (i.e., at 9.5 weeks old of mouse) (see FIG. 1).

2. Immunofluorescence

(31) After immobilization of the mouse's cerebrum and hippocampus, 0.5% thioflavin S (Sigma-Aldrich), anti-20G10 against Aβ42 (mouse, 1:1000) and anti-G30 against A1340 (rabbit, 1:1000), anti-GFAP (rabbit, 1:500, DAKO) and anti-Iba-1 (rabbit, 1:500, WAKO) were incubated together. The sites were analyzed using a confocal laser scanning microscope or an Olympus BX51 microscope equipped with Fluoview SV1000 imaging software (Olympus FV1000, Japan). A percentage of an area of a stained area to an area of total tissues was quantified and analyzed by using Metamorph software (Molecular Devices).

3. Real-Time Quantitative PCR

(32) A real-time quantitative PCR method was used to measure the expression levels of inflammatory response-related cytokines (TNF-α, IL-1β, and IL-6). Total RNA was extracted from the brain tissue using an RNeasy Plus mini kit (Qiagen, Korea, Ltd), and cDNA was synthesized from a total of 5 μg of RNA using a kit from Clontech (Mountain View, Calif.). In addition, by using a Corbett research RG-6000 real-time PCR instrument, real-time quantitative PCR was performed by setting 95° C., 10 minutes; 95° C., 10 seconds; and 58° C., 15 seconds as one cycle and repeating 40 cycles. Primer pairs used in the real-time quantitative PCR are shown in Table 1.

(33) TABLE-US-00001 TABLE 1 mTNF-α Forward 5′-GAT TAT GGC TCA SEQ ID NO: 1: GGG TCC AA-3′ Reverse 5′-GCT CCA GTG AAT SEQ ID NO: 2: TCG GAA AG-3′ mIL-1β Forward 5′-CCC AAG CAA TAC SEQ ID NO: 3: CCA AAG AA-3′ Reverse 5′-GCT TGT GCT CTG SEQ ID NO: 4: CTT GTG AG-3′ mIL-6 Forward 5′-CCG GAG AGG AGA SEQ ID NO: 5: CTT CAC AG-3′ Reverse 5′-TTG CCA TTG CAC SEQ ID NO: 6: AAC TCT TT-3′ mGAPDH Forward 5′-TGA ATA CGG CTA SEQ ID NO: 7: CAG CAA CA-3′ Reverse 5′-AGG CCC CTC CTG SEQ ID NO: 8: TTA TTA TG-3′

4. Western Blot

(34) Expression of the following genes was analyzed using Western blotting. First, antibodies against LC3, beclin-1, and p62 [all, purchased from cell signaling Technologies], cathepsin D (R&D systems) and β-actin (Santa Cruz) were used, and density quantification was performed by using ImageJ software (US National Institutes of Health).

5. Behavioral Experiment

(35) In order to confirm potential effects on learning and memory, Morris water maze (MWM) and fear conditioning tests were performed. In the MWM, the mouse learned a task 4 times a day for 10 days, a platform was removed on day 11, and a probe trial was performed. In the fear conditioning, on the first day, the mouse was placed in a conditioning chamber, and sound stimulation (10 kHz, 70 dB) and electrical stimulation (0.3 mA, 1 s) were given. On the second day, the memory on a space was confirmed without stimulation in the same conditioning chamber as the first day, and on the third day, the memory test for fear was performed when only the sound stimulation was given in another conditioning chamber.

6. Statistical Analysis

(36) For comparison of two groups, a T-test of a student was performed, while for comparison of multiple groups, repeated measurement analysis of a Tukey's HSD test and a variance test was performed according to an SAS statistical package (release 9.1; SAS Institute Inc., Cary, N.C.). *p<0.05 and **p<0.01 were considered to be significant.

Experimental Results

1. Confirmation of Reduction of Amyloid-β Deposition in APP/PS1 Mouse Injected with Autophagy Enhancing Compound

(37) In order to determine whether the aforementioned autophagy enhancing compound had effects for prevention and treatment on neurodegenerative disease, the effect of the compound on an Alzheimer's model was representatively evaluated. Among the autophagy enhancing compounds obtained in Example 1, 5-methoxy-4-(phenylsulfonyl)-2-(4-(trifluoromethyl)phenyl)oxazole (Example 1-1) was typically used, and in the same manner as shown in FIG. 1, the autophagy enhancing compound was administered intraperitoneally to an Alzheimer's animal model for 2 months and the results were evaluated.

(38) First, an amyloid-β (Aβ) profile of an Alzheimer's lesion and the deposition degree of tau protein were confirmed. First, the cerebral cortex and hippocampus regions of the mouse were stained with thioflavin S (ThioS) according to a known method to confirm the deposition degree of fibrillar amyloid-β. In addition, immunofluorescence staining of Aβ40, Aβ42, and AT8 was performed to confirm the deposition degree of amyloid-β and tau protein.

(39) As a result of the experiment, compared to the APP/PS1 mouse, the deposition of fibrillar Aβ (see FIG. 2) and the deposition of Aβ40 and Aβ42 (see FIGS. 3A and 3B) and tau protein (see FIG. 4) in an APP/PS1 mouse injected with the autophagy enhancing compound were confirmed to be significantly low.

2. Confirmation of Improvement of Learning and Cognition in APP/PS1 Mouse Injected with Autophagy Enhancing Compound

(40) To determine potential effects of an autophagy enhancing compound on learning and cognition in an Alzheimer's animal, Morris water maze (MWM) and fear conditioning tests were performed.

(41) As illustrated in FIGS. 5A to 5C, the APP/PS1 mouse showed severe impairment in spatial memory, cognition and memory formation, but it was confirmed that in the APP/PS1 mouse injected with the autophagy enhancing compound, such impairment was significantly improved (FIGS. 5A to 5C). In addition, it was confirmed that the autophagy enhancing compound showed a remarkable memory improvement effect even in the fear conditioning test (FIG. 5D).

3. Confirmation of Neuroinflammatory Change in APP/PS1 Mouse Injected with Autophagy Enhancing Compound

(42) In order to confirm an effect of the injection of the autophagy enhancing compound on a neuroinflammatory change in an Alzheimer's animal, the present inventors observed changes in astrocytes (using GFAP as a marker) and microglia (using Iba-1 as a marker) in the brain.

(43) As a result of the experiment, compared with the APP/PS1 mouse, it was confirmed that the activities of astrocytes and microglia were significantly reduced in the APP/PS1 mouse injected with the autophagy enhancing compound (FIGS. 6A and 6B). In addition, in the APP/PS1 mouse, the gene expression of inflammatory cytokines TNF-α, IL-1β and IL-6 was significantly increased compared to a wild mouse, but in the APP/PS1 mouse injected with the autophagy enhancing compound, it was confirmed that the expression of the inflammatory cytokines was restored to a normal level (FIG. 6C). Through these results, it was confirmed that the injection of the autophagy enhancing compound modulated a neuroinflammatory response in an Alzheimer's brain environment.

4. Confirmation of Effect on Autophagy-Related Genes in APP/PS1 Mouse Injected with Autophagy Enhancing Compound

(44) In order to confirm how the aforementioned autophagy enhancing compound (in particular, 5-methoxy-4-(phenylsulfonyl)-2-(4-(trifluoromethyl)phenyl)oxazole) actually act in an autophagy-related pathway in vivo, conversion of LC3-I to LC3-II, and expression levels of beclin-1, cathepsin D and p62 were confirmed through a Western blotting experiment in brain tissue samples of 9.5-month-old WT, APP/PS1 (untreated group), and an APP/PS1 mouse injected with the autophagy enhancing compound.

(45) The autophagy occurs through a fusion process of autophagosomes and lysosomes. In the previous study (Korean Patent Registration No. 10-1521117), the present inventors identified that an ideal change in turnover of autophagic vacuoles (AV) is shown in a neurodegenerative disease state such as Alzheimer's disease and abnormal changes occur in which autophagosomes are not degraded but continuously accumulated. As illustrated in FIG. 7, compared to the wild-type (WT) mouse, in the APP/PS1 mouse (untreated), it was confirmed an abnormal change in which the level of LC3-II was increased. On the other hand, it was confirmed that in the APP/PS1 mouse injected with the autophagy enhancing compound, the level of LC3-II was decreased to a level similar to WT, and it is meant that the degradation of autophagic vacuoles was well caused by the compound. It was confirmed that the expression of beclin-1 had no large difference in the three groups.

(46) In addition, the expression of cathepsin D (lysosomal hydrolase) and p62, which are indicators of autophagy turnover, is increased in Alzheimer's patients and pathologically related to Alzheimer's disease. Compared to the WT mouse, it was confirmed that the expression levels of cathepsin D and p62 were increased in the APP/PS1 mouse, and the increased expression levels of cathepsin D and p62 were decreased in the APP/PS1 mouse injected with the autophagy enhancing compound.

(47) Summarizing the above results, it could be seen that the injection of the autophagy enhancing compound in the APP/PS1 mouse reduced Aβ plaque deposition and inflammatory response, and restored damaged autophagy. In addition, it could be seen that the autophagy enhancing compound can be used as a therapeutic agent for degenerative brain disease including Alzheimer's disease by improving learning and memory in an Alzheimer's animal.

INDUSTRIAL AVAILABILITY

(48) As described above, the present invention relates to a novel phenylsulfonyl oxazole derivative and a use thereof and specifically, to a compound represented by Chemical Formula 1 in the present specification or a pharmaceutically acceptable salt thereof, and to a use thereof for prevention, treatment, or improvement of neurodegenerative disease.

(49) The compound of the present invention has a remarkable therapeutic effect, such as reduction of Aβ plaques, alleviation of neuroinflammation, and improvement in memory and anxiety, by regulating abnormal autophagy when applied to neurodegenerative disease such as Alzheimer's disease. Therefore, the compounds of the present application can be very useful for the development of agents for prevention or treatment of neurodegenerative diseases, and thus have great industrial availability.