KR20090002888A - Pharmaceutical composition comprising substituted benzene analog compound with neuroprotective activities for the treatment of neurological brain disease comprising parkinson's disease and method for treating brain disorder using the compounmd - Google Patents

Abstract

A pharmaceutical composition for treating the cranial nervous disease, and a method for treating the cranial nervous disease by using the composition are provided to improve the effect for treating the cranial nervous disease such as Parkinsons disease, stroma, depression, pain, anxiety and the degenerative brain disease. A pharmaceutical composition for treating the cranial nervous disease comprises a substituted benzene derivative compound represented by the formula I or its pharmaceutically acceptable salt; and a carrier, wherein R is a substituted or unsubstituted aryl or heteroaryl group; Y1 and Y2 are independently H or a C1-C3 alkyl group; A is N, O, S, C-OR', C=O, C(O)NH, NHC(O), CH2, CH=CH, or OCH2C(OR')H; R1 is H or a C1-C5 substituted or unsubstituted alkyl group; B is a cyclic aromatic amine group, a guanidine group or a heterocyclic aliphatic amine group; X is H or a halogen atom; m is an integer of 0-4; and n is an integer of 0-5.

Description

Pharmaceutical composition comprising a substituted benzene derivative compound having a therapeutic effect and neuroprotective effect on cerebral neurological diseases including Parkinson's disease and a method for treating brain diseases using the compound NEUROLOGICAL BRAIN DISEASE COMPRISING PARKINSON'S DISEASE AND METHOD FOR TREATING BRAIN DISORDER USING THE COMPOUNMD}

The present invention provides a substituted benzene derivative compound having a therapeutic effect on a neurological disorder including Parkinson's Disease and a therapeutic effect on epilepsy, depression, pain, anxiety or degenerative brain disease, a pharmaceutical composition comprising an effective amount thereof, and the compound The present invention relates to a method for treating cerebral neurological diseases, including Parkinson's disease, epilepsy, depression, pain, anxiety or degenerative brain diseases, by administering an effective amount to a mammal.

Parkinson's disease is the second most common degenerative neurological disease. It is a refractory disease that is becoming a social and economic problem due to the increasing incidence of the elderly population. About 4 million people are known to have this disease worldwide, and the number of patients in the United States is increasing by 50,000 each year. The incidence rate is one in 1,000, and the higher the age, the more frequent the occurrence. The clinical features of the disease include dyskinesia, cognitive dysfunction, and depression (Resting tremor, Bradykinesia, Rigidity, Postural instability). There are non-motor abnormalities accompanied by depression, sleep disorders, and pain. This disease is known to be the most associated with aging, and environmental factors, free radicals and genetic factors (5-10%) caused by neurotoxins such as pesticides are known to affect the onset. The cause of the disease is unknown. Genetic factors are known to be associated with gene mutations such as alpha-synuclein, Parkin, PINK-1, UCH-L1, DJ-1 and the like.

Anatomically, Parkinson's disease is associated with extensive degeneration of the dopaminergic neurons (substantia Nigra) located in the basal ganglia of the brain. When about 60-80% of the dopamine produced by the neurons is damaged, the extraramidal tract line can no longer effectively promote movement, resulting in Parkinson's disease symptoms.

Parkinson's disease is not known for its exact etiology, so treatment is mainly used to improve symptoms rather than radical treatment. Therapies currently in use or under development include: The most developed and used drugs include dopamine precursors such as levodopa, a dopamine supplement, and dopamine receptor agonists (fenofibrate). In addition, COMT inhibitors and MAO-B (monoamine oxidase B) inhibitors, which inhibit dopamine metabolism and maintain the concentration of dopamine in the brain, are used. Anti-muscarinics and NMDA antagonists have been developed and used as neurotransmitters to improve neurotransmitters other than dopamine. Cerebral nerve cell protective agents, antioxidants, inhibitors of brain cell death, and anti-brain function are used as treatments. Efforts are ongoing. Terminally ill patients who are no longer effective with pharmacotherapy are applying surgical treatments such as deep brain stimulation.

MAO-B not only plays an important role in maintaining dopamine concentrations, but also plays an important role in the control of brain cell damage, and various evidences suggest that MAO-B inhibitors are likely to have neuroprotective effects. have. MAO-B plays an important role in the pathogenesis of Parkinson's disease caused by MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) or similar environmental toxicants. Inhibition will act to inhibit the development of Parkinson's disease. In addition, toxic oxygens are generated during the metabolism of dopamine by MAO-B, and by inhibiting the activity of MAO-B, they will inhibit the production of dopamine neurons by inhibiting their production. MAO-B inhibitors have a variety of neuroprotective effects that other therapeutic agents do not have, suggesting a variety of evidence from experimental and animal and clinical evidence at the cellular level. In particular, the administration of MAO-B inhibitors early in the onset may have the effect of preventing the progression of Parkinson's disease, and may reduce the wear-off effect of administration of dopamine precursors such as levodopa, levodopa or It has the advantage of delaying the administration of dopamine receptor agonists. In addition, it may play a role in prolonging the effect of levodopa to reduce the behavioral symptoms by prolonging the action time. In particular, MAO-B inhibitors have the advantage that they can be used in patients from the early stage to the late stage, unlike other therapeutic agents.

In recent years, therapeutics are being developed based on the increased MAO-B activity in the brains of Alzheimer's disease patients, and there are active attempts to apply them to stroke, head trauma and other degenerative brain diseases. ought.

Selegiline (Deprenyl), a MAO-B inhibitor, is used as a gold standard for Parkinson's disease, but its use is limited due to hepatotoxicity and its metabolites, metamphetamine. There are a lot. However, a new MAO-B inhibitor, Azilect (Rasagiline), which was first released in Europe in 2005 and was approved by the US FDA in 2006, appeared, which is a new drug for Parkinson's disease that supplements the disadvantages of selegiline. It began to be known as. The value of the new drug could be even greater if it is supported by clinical trials that Agilent has a neuroprotective effect that existing drugs do not have.

However, because selegiline and lasazilin are irreversible MAO-B inhibitors, they can inhibit MAO-B activity until new MAO-B is made in vivo, but this increases the likelihood of unpredictable side effects. As an alternative to compensate for such drawbacks, if the enzyme exhibits a potent enzyme inhibitory activity while reversibly inhibiting the activity, it is expected to be superior in terms of safety or efficacy than conventional irreversible inhibitors. Recently, a reversible MAO-B inhibitor called Safinamide has been developed and is in clinical trials (Phase III), but no remarkable reversible MAO-B inhibitor has yet been developed.

In addition to dyskinesia in Parkinson's disease, the most common non-motor disorders are depression, pain, cognitive dysfunction, and sleep disorders. More than 40% of Parkinson's patients develop depressive symptoms and are more likely to have Parkinson's disease and are associated with damage to anatomically similar locations (Orbital cortex and basal ganglia). Simultaneous administration of antidepressants (SSRI, SNRI, TCA) and Parkinson's disease treatments has been reported to exacerbate symptoms due to drug interactions. Pain symptoms also occur in 45-70% of Parkinson's patients and are thought to occur with lowered pain detection thresholds. Leuk bodies, such as those found in amygdala in the brain of Parkinson's disease patients, are thought to be associated with anxiety symptoms.

Therefore, if a single drug can act on the above non-motor dyskinesias in addition to the therapeutic effect of dyskinesia, it is expected to have a great advantage in terms of efficacy and dosage.

An object of the present invention is a Parkinson's disease, epilepsy, depression, pain, which contains an effective amount of a substituted benzene derivative compound and a pharmaceutically acceptable salt thereof having a therapeutic effect and brain protective effect of cerebral neurological diseases including Parkinson's disease, The present invention provides a pharmaceutical composition for treating cranial nerve disease, including anxiety and degenerative brain disease.

Another object of the present invention is to administer a pharmaceutical composition comprising the substituted benzene derivative compound to a mammal to treat cerebral neurological diseases including Parkinson's disease, epilepsy, depression, pain, anxiety and degenerative brain diseases. There is.

According to one aspect of the invention, Parkinson's disease, epilepsy, depression, pain, anxiety and degenerative brain, comprising an effective amount of a substituted benzene derivative compound of formula (I) and pharmaceutically acceptable salts thereof Provided are pharmaceutical compositions for treating neurological diseases, including diseases.

Formula (I)

According to another aspect of the present invention, there is provided a method for treating neurological diseases, including Parkinson's disease, epilepsy, depression, pain, anxiety and degenerative brain diseases, characterized by administering an effective amount of the substituted benzene derivative compound to a mammal. Is provided.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.

The present invention relates to a pharmaceutical composition for treating neurological diseases, comprising an effective amount of a substituted benzene derivative compound represented by formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. :

Formula (I)

Wherein R is selected from the group consisting of substituted or unsubstituted aryl including benzene, and heteroaryl including thiazole, oxazole, imidazole, pyridine, wherein aryl and heteroaryl are -F, -Cl, halogen elements, such as -Br, alkyl of C _{1 ~ C 6, C 1 ~} C 6 alkoxy, -CF _3, and may be substituted with one or more substituents selected from the group consisting of -OCF _3, preferably from Ph-, (2-Cl-5-F-Ph)-, (3-F-Ph)-, (2,6-di-F-Ph)-, (2,4-di-F-Ph)-, (3 -MeO-Ph)-, (2,4-di-F-Ph)-, 4-pyridyl-, toluene, 1- (trifluoromethyl) benzene and 1- (trifluoromethoxy) benzene Selected from the group;

Y ₁ and Y ₂ are independently selected from the group consisting of H and C ₁ -C ₃ alkyl;

A is N, O, S, C-OR ', C = O, C (O) NH, NHC (O), CH ₂ , CH = CH and OCH ₂ C (OR') H where R 'is H or C ₁ to C ₅ substituted or unsubstituted alkyl group), preferably O, C═O, C—OH, NHC (O), CH ₂ , OCH ₂ CHOH and CH═ Is selected from the group consisting of CH;

B is selected from the group consisting of cyclic aromatic amines including imidazole, triazole and tetrazole, and heterocyclic aliphatic amines including guanidine, pyrrolidine, piperidine and morpholine, preferably Imidazole, triazole, tetrazole, guanidine, pyrrolidine, piperidine and morpholine;

X is selected from the group consisting of H and halogens including fluorine, chlorine and bromine, preferably H or fluorine;

m is an integer of 0-4, Preferably it is an integer of 0-2;

n is an integer of 0-5, Preferably it is an integer of 0-3.

These compounds and their pharmaceutically useful salts are involved in the action of Na ⁺ channels, dopamine transporters, norepinephrine transporters and serotonin receptors, including MAO-B activity inhibitory effects, thereby treating Parkinson's disease and protecting brain cells. It is also effective in the treatment of neurological diseases including epilepsy, depression, pain and anxiety.

In the present invention, the compounds may be chemically synthesized by the reaction shown below. However, this is merely exemplary and the present invention is not limited thereto, and may be synthesized by other reactions known to those skilled in the art.

Scheme 1

As in Scheme 1, 4- (benzyloxy) phenol and 1-bromo-2-chloroethane were stirred under reflux for 12 hours with acetonitrile as a solvent to obtain 1-((4- (2-chloroethoxy) phenoxy). Methyl) benzene was synthesized and 1- (2- (4-benzyloxy-phenoxy) ethyl) -1H-imidazole was prepared by stirring and refluxing acetonitrile with a solvent under the basic conditions of imidazole and K ₂ CO ₃ . can do.

Scheme 2

As in Scheme 2, 1- (2- (4- (benzyloxy) phenoxy) ethyl) -1H- was hydrogenated under Pd / C to give 4- (2- (1H-imidazol-1-yl). Methoxy) phenol was synthesized, and acetonitrile was refluxed with a solvent under the basic conditions of _3- fluorobenzyl bromide and K ₂ CO ₃ to obtain 1- (2- (4- (3-fluorobenzyloxy) phenoxy) Ethyl) -1H-imidazole was prepared.

Representative examples of the compound of formula (I) are as follows, but these are merely illustrative and the present invention is not limited thereto.

Compound 1

1- [2- (4-benzyloxy-phenoxy) -ethyl] -1 H-imidazole

Compound 2

1- {2- [4- (2-Chloro-5-fluoro-benzyloxy) -phenoxy] -ethyl} -1 H-imidazole

Compound 3

1- {2- [4- (3-Fluoro-benzyloxy) -phenoxy] -ethyl} -1 H-imidazole

Compound 4

1- {2- [4- (2,6-Difluoro-benzyloxy) -phenoxy] -ethyl} -1 H-imidazole

Compound 5

1- {2- [4- (2,4-Difluoro-benzyloxy) -phenoxy] -ethyl} -1 H-imidazole

Compound 6

1- [3- (4-benzyloxy-phenoxy) -propyl] -1 H-imidazole

Compound 7

1- (4-benzyloxy-phenyl) -3-imidazol-1-yl-propan-1-one

Compound 8

1- (4-benzyloxy-phenyl) -2- [1,2,3] triazol-1-yl-ethanol

Compound 9

1- [4- (3-methoxy-benzyloxy) -phenyl] -2-tetrazol-2-yl-ethanol

Compound 10

1- {2- [4- (2,4-Difluoro-benzyloxy) -2-fluoro-phenyloxy] -ethyl} -1 H-imidazole

Compound 11

4- [4- (2-imidazol-1-yl-ethoxy) -phenoxymethyl] pyridine

Compound 12

N- (4-benzyloxy-phenyl) -2-imidazol-1-yl-acetamide

Compound 13

1- [2- (4-benzyloxy-phenoxy) -ethyl] -pyrrolidine

Compound 14

N- [2- (4-benzyloxy-phenoxy) -ethyl] -guanidine

Compound 15

1- [3- (4-benzyloxy-phenyl) -propyl] -pyrrolidine

Compound 16

1- (4-benzyloxy-phenoxy) -3-imidazol-1-yl-propan-2-ol

Compound 17

1-[(E) -3- (4-benzyloxy-phenyl) -allyl] -1 H-imidazole

Compound 18

1- {2- [4- (2-Methyl-benzyloxy) -phenoxy] -ethyl} -1 H-imidazole

Compound 19

1- {2- [4- (3-Trifluoromethyl-benzyloxy) -phenoxy] -ethyl} -1 H-imidazole

Compound 20

1- {2- [4- (3-Trifluoromethoxy-benzyloxy) -phenoxy] -ethyl} -1 H-imidazole

Compound 21

1- {2- [4- (1-phenyl-ethoxy) -phenoxy] -ethyl} -1 H-imidazole

 1- [2- (4-benzyloxy-phenoxy) -ethyl] -1H-imidazole, represented by Compound 1, is made during the synthesis of the β1-Selective Adrenoceptor antagonist. (Machin PJ et al., (1984). Β1-Selective Adrenoceptor Antagonists: 4-Azolyl-Linked Phenoxypropalamines potent with β1-blockade, J. Med. Chem. 27, 503-509)

Benzene derivative compounds of formula (I) and their pharmaceutically useful salts are involved in the action of Na ⁺ channel, dopamine transporter, norepinephrine transporter and serotonin receptors, including MAO-B activity inhibitory effect, Parkinson's disease It has a therapeutic effect and a protective effect on brain cells, and is effective in treating neurological diseases including epilepsy, depression, pain, anxiety or degenerative brain diseases. Accordingly, the compound of formula (I) may be used alone or in combination with a pharmaceutically acceptable carrier to treat cerebral neurological diseases, including Parkinson's disease, epilepsy, depression, pain, anxiety, or degenerative brain diseases. .

Pharmaceutical compositions can be administered by oral, rectal, nasal, lung, topical, transdermal, intracisternal, intraperitoneal, vaginal and parenteral (subcutaneous, intramuscular, intrathecal, intravenous and intradermal) routes It may be specifically formulated to be administered by any suitable route such as, preferably by oral route. Preferred routes will depend on the general conditions and age of the subject being treated, the nature of the condition being treated and the active ingredient selected.

The pharmaceutical compositions prepared according to the invention may be in any suitable route, for example in tablets, capsules, powders, granules, pellets, oral tablets, dragees, pills or tablets, aqueous or non-aqueous liquids. Parenteral administration in the form of oral or injectable solutions in the form of aqueous solutions or suspensions, or oil-in-water or water-in-oil liquid emulsions, elixirs, syrups and the like. Other pharmaceutical compositions for parenteral administration include sterile powders composed of sterile injectable solutions or dispersions prior to use as well as dispersions, suspensions or emulsions. Depot injection formulations are also considered to be within the scope of the present invention. Other suitable dosage forms include suppositories, sprays, ointments, creams, gels, inhalants, skin patches, and the like. Any method known in the art may be used to prepare the composition, and any pharmaceutically acceptable carrier diluent, excipient or other additive commonly used in the art may be used.

The present invention also provides a method for the treatment of cerebral neurological diseases, including Parkinson's disease, epilepsy, depression, pain, anxiety or degenerative brain diseases, characterized in that the substituted benzene derivative compound is administered to a mammal in an effective amount.

Typically the pharmaceutical compositions of the present invention are administered in unit dosage form containing the active ingredient in an amount of about 0.5 to 2 mg. The total daily dose is usually about 0.5 to 2 mg, preferably 1 to 2 mg of the active compound of the present invention, but falls within the range in view of the patient's situation and also taking into account the activity of the administered drug. Certain amounts may be administered that do not. Determining the optimal dose for a particular situation should be determined experimentally.

The compound of the present invention may be administered in one or several doses, and preferably, the daily dose is divided once or twice daily. It may be administered alone or in combination with a pharmaceutically acceptable carrier or excipient. The pharmaceutical compositions according to the invention may be formulated according to the prior art with any other known auxiliaries and excipients as well as pharmaceutically acceptable carriers or diluents. The formulations may conveniently be presented in unit dosage form by methods known in the art of pharmacy.

Such carriers are conventionally used in the preparation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrroli Don, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil, and the like.

In order to verify the Parkinson's disease treatment effect of the representative compounds of the formula (I) of the present invention, behavioral change analysis using brain MAO-B inhibition, MPTP mouse model and 6-OHDA rat model, Parkinson's disease animal model, brain Tissue lesion analysis and dopamine concentration analysis were performed, and the efficacy of each disease animal model was measured to verify the effects of treating epilepsy, depression, pain and anxiety.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

The compound of formula (I) of the present invention can inhibit the progression of Parkinson's disease as well as the treatment of behavioral disorders that accompany brain dysfunction in Parkinson's disease patients by inhibiting MAO-B activity and inhibiting brain cell damage. It has a prophylactic effect and also shows therapeutic efficacy in epilepsy, depression, pain, and anxiety animal models, so that the pharmaceutical composition containing the same can be usefully used as a medicament for the treatment of related diseases.

Example  One: Monoamine oxidase ( Monoamine oxidase ) ( MAO -B) activity inhibitory effect

1) Materials and Methods

100 μL of enzyme buffer prepared by diluting 1/200 of 5 mg / mL monoamine oxidase A or B human-derived enzyme with 0.05 M sodium phosphate (pH 7.4) buffer and adding 2 μL of the solution of Compound 1 at the corresponding concentration. Place on bottom plate and incubate for 30 minutes.

400 μM Amplex Red agonist, 2 U / mL Horseradish peroxidase, 2 mM substrate (tyramine for MAO-A, benzylamine for MAO-B in 0.05M sodium phosphate, pH 7.4 buffer 100 uL of working buffer made of)) is mixed 1: 1 with pre-incubated enzyme buffer and measured by fluorescence (EX: 563 nm & EM: 587 nm) for 30 minutes.

2) results

Compound 1 inhibited 31.7% of MAO-A at 10μM and had a value of (IC ₅₀ ) of 0.011μM that inhibited MAO-B activity by 50%. Compounds 2, 3 and 4 were also carried out in the same manner as described above to show the inhibitory effect on MAO-A and MAO-B. All of them showed potent inhibitory effect on MAO-B while little inhibitory effect on MAO-A.

Table 1.Inhibitory Effects on MAO-A and MAO-B Activity Derived from Rat Brain (μM)

Compound number One 2 3 4 MAO-A ~ 100 μM 1.1 μM 9.9 μM 10.1 μM MAO-B 0.011 μM 0.068 μM - 0.033 μM

In addition, more specific inhibitory effects on MAO-B derived from rat brain and human brain on the total compounds are shown below (Table 2.).

Table 2. Inhibitory Effects on the Activity of MAO-B

IC50 value or concentration MAO-B derived from the rat brain MAO-B derived from the human brain Compound 1 IC ₅₀ 11.2 nM 16.2 nM Compound 2 IC ₅₀ 68.3 nM Compound 3 IC ₅₀ 17.6 nM Compound 4 IC ₅₀ 32.7 nM 6.23 nM Compound 5 1 μM 86.63% Compound 6 10 μM 86.10% Compound 7 IC ₅₀ 96.9 nM Compound 8 1 μM 71.70% Compound 9 1 μM 76.80% Compound 10 1 μM 99.63% Compound 11 1 μM 58.35% Compound 12 1 μM 97.64% Compound 13 1 μM 85.70% Compound 14 1 μM 83.82% Compound 15 1 μM 80.57% Compound 16 1 μM 72.48% Compound 17 1 μM 95.20% Compound 18 1 μM 97.42% Compound 19 1 μM 86.5% Compound 20 1 μM 36.12% Compound 21 1 μM 90.64%

 As shown in Table 2, the compounds 1,2,3,4 and 7 have a very strong inhibitory effect of MAO-B since the IC50 values are 11.2 nM to 96.9 nM, and the remaining compounds are also 72.48% at 1 μM. The inhibitory effect of MAO-B up to 97.64% was shown to be very strong.

The potent inhibitory effect of MAO-B activity of the substituted benzene derivative compounds identified in the present invention demonstrates their availability as a therapeutic agent for Parkinson's disease.

Example  2 : MPTP Caused by Parkinson's disease  Identify effects on mouse models

Protective Effect of Substituted Benzene Derivative Compounds Against Dopamine Neuron Damage in Parkinson's Disease Animal Model by Administering MPTP (1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine) It was confirmed.

A commonly used MPTP acute dose model consists of four doses of 15-25 mg / kg MPTP (free base), two times a day, per day (T. Breidert et al., (2002) Protective action of the peroxisome proliferators-activated receptor-r agonist piolitazone in a mouse model of Parkinson's disease.J neurochem. 82: 615-624) Three days after MPTP treatment, dopamine neurons were markedly killed and dopamine content in the striatum was 70-80 compared to the control group. % Is reported to decrease gradually after 7-8 days of MPTP treatment (Bezard E et al., (2000) Adaptive changes in the nigrostriatal pathway in response to increased 1-methyl-4-phenyl-1,2 , 3,6-tetrahydropyridine-induced neurodegeneration in the mouse.Eur J Neurosci. 12 (8): 2892-2900; Khaldy H et al., (2003) Synergistic effects of melatonin and deprenyl against MPTP-induced mitochondrial damage and DA depletion Neurobiol Aging. 24 (3): 491-500; Y. Muramatsu et al., (2002) Therapeutic effect of neuronal nitric oxide synthase inhibitor (7-Nitroindazole) against MPTP neurotoxicity in mice.Metabolic Brain Dis. 17 (3): 169-182).

1) Materials and Methods

end. MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) and drug treatment

Models were constructed by intraperitoneally administering MPTP (20 mg / kg, free base; Sigma, St. louis, MO) three times per day to 8-week-old (20-25 g) male C57BL / 6 assay mice. (Breidert et al., 2002; Lichuan Yang, Shuei Sugama, Jason W. Chirichigno, Jason Gregorio, Stefan Lorenzl, Dong H. Shin, Susan E. Browne, Yoshinori Shimizu, Ton H. Joh, M. Flint Beal, 1and David S. Alber. (2003) Minocycline Enhances MPTP Toxicity to Dopaminergic Neurons.J Neurosci Res. 74: 278-285). To see the protective effect of candidate compound 1, it was dissolved in 30% polyethylene glycol (PEG) and administered intraperitoneally 30 minutes before and 30 minutes after MPTP treatment at doses of 10 mg / kg, 20 mg / kg and 40 mg / kg. It was. The control group without administration of MPTP (Sham) was made by intraperitoneally administering saline instead of MPTP and intraperitoneally with 30% PEG 30 minutes before and after PBS treatment.

I. Immunohistochemical Analysis

Changes in TH expression in striatum and medulla following MPTP and drug administration were measured using immunohistochemical staining. Seven days after drug administration, the animals were anesthetized with chloral hydrate (300 mg / kg), the rib cage was opened, and 200 ml of 0.1 M PBS (pH 7.4) was perfused into the heart to remove blood from the blood vessels. After sufficient blood was removed, 250-300 ml of 4% paraformaldehyde / PBS fixative was perfused and the brain was extracted and postfixed in the refrigerator for 24 hours with the same fixative. The brain tissues were washed with PBS, and then transferred to 30% sucrose solution to prevent ice crystals from freezing and stored until the tissues subsided, embedded in OCT compound and frozen at -20 ℃. Subsequently, a continuous coronal section was performed to a thickness of 40 μm using the cryostat (Reichert Frigocut model 2000). The stored tissues were reacted in 3% H ₂ O ₂ / PBS for 30 minutes and then in 0.1 M PBS containing 0.3% Triton X-100 and 3% bovine serum albumin. For selective staining of dopamine cells, primary antibodies were reacted overnight at room temperature using anti-mouse monoclonal TH (Chemicon International, Temecula, CA; 1: 500) and biotinylated goats as secondary antibodies. (goat) -anti-mouse IgG (Vector Lab, Burlingame, CA, 1: 200) was used. Avidin-biotin binding was induced using the Vectastain elite ABC kit (Vector Lab, Burlingame, Calif.) And then developed using 3,4-diaminobenzidine (DAB).

All. Determination of Dopamine and its Metabolites in Striatum

Changes in the content of dopamine and dopamine metabolites in striatum with MPTP and drug administration were measured using high performance liquid chromatography (HPLC). Seven days after the administration of the medicine, the animals were sacrificed by cervical distal bone and brain tissues were immediately extracted. A striatum was obtained from the extracted brain tissue, and cold 0.5 ml HPLC analysis solution (0.1 M perchloric acid, 0.1 mM EDTA) was added, and then tissue homogenates were prepared using an ultrasonic grinding machine. This was centrifuged at 12,000 rpm for 15 minutes and the supernatant was filtered through a nitrocellulose membrane filter (0.2 μM, Millipore). HPLC analysis used uBondapakTM C18 column (4.6 x 150 mm, particle size 10 μM: Shisheido, Japan) and mobile phase (0.07 M monobasic sodium phosphate, 1 mM sodium octasulfonic acid, 0.1 μM EDTA, 5% aceto Nitrile, pH 3.2), the flow rate was maintained at 0.7 ml / min and the electrode potential of the electrochemical detector (ICA-5000, Japan) was 700mV.

la. Statistical analysis

Five or more experimental results were used to confirm the damage and protective effect of dopamine neurons following MPTP and drug administration. The experimental data were expressed as mean ± standard error mean (SEM). Statistical analysis was tested by Student's t-test after one-way ANOVA test, and was determined to be significant when p value was 0.05 or less.

2) results

end. Changes in Dopamine and Their Metabolites in Striatum

In order to examine the protective effect of the drug against MPTP toxicity, changes in the dopamine and metabolites in the striatum were measured at 7 days after MPTP administration.

Dopamine content of 1045.3 ± 151.2 pmole / mg protein and 1018.5 ± 238.3 pmole / mg protein, respectively, was not significantly changed in the control group treated with Compound 1 (40 mg / kg). After MPTP treatment, dopamine content in striatum was reduced to 41.5% (372.8 ± 67.6 pmole / mg protein) of control group (898.8 ± 95.9 pmole / mg protein). However, when 10, 20 and 40 mg / kg of Compound 1 were administered, 69.6% (625.8 ± 64.2 pmole / mg protein), 86.7% (779.4 ± 47.7 pmole / mg protein) and 90.7% (815.5 ± 60.1 pmole) of the control group, respectively. / mg protein) showed a concentration-dependent recovery.

On the other hand, the amount of DOPAC, a dopamine metabolite, was reduced to 58.6% (35.0 ± 6.7 pmole / mg protein) of the control group (59.7 ± 8.2 pmole / mg protein) in the MPTP treatment group. When 20 mg / kg was administered, the DOPAC content was recovered to 93.2% (55.6 ± 12.7 pmole / mg protein) of the control group, but when 40 mg / kg was administered, the DOPAC content was 62.9% (37.5 ± 5.2 pmole / mg protein). ), There was no difference compared to the MPTP treatment group. The content of norepinephrine tended to decrease slightly by compound 1 but did not show statistical significance (FIG. 1).

Figure 1 shows the concentration of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and nor-epinephrine (NE) in striata using HPLC-ECD 7 days after MPTP administration One result is shown. DA concentration was significantly decreased in the MPTP alone group, but the dopamine concentration of the control group was maintained in the compound 1 administration group. This value is mean ± SEM (n = 8).

* Is p <0.05 compared to control, # is p <0.05 compared to MPTP alone group.

Dopamine turnover by MPTP (DA turnover = DA / DOPAC + HVA) was reduced to 63.0% (3.8 ± 0.5) of the control (6.0 ± 0.1) and 112.7% (6.8) of the control by 20 mg / kg of compound 1, respectively. ± 0.2) and 103.0% (6.2 ± 0.3). However, administration of 40 mg / kg Compound 1 increased dopamine turnover to 165.0% (9.9 ± 0.2) of the control group (FIG. 2).

In FIG. 2, the DA turnover rate is expressed by the formula of DA / (DOPAC + HVA), which was significantly decreased in the MPTP-only group, whereas the compound 1-treated group was similar to the sham group. This value is mean ± SEM (n = 8).

* Is p <0.05 compared to control, # is p <0.05 compared to MPTP alone group.

A. Control, B. MPTP alone group, C. MPTP + 1 compound (10mg / kg), D.MPTP + 1 compound (20mg / kg), E. MPTP + 1 compound (40mg / kg)

The dopamine concentration retention effect on Compounds 2, 3 and 4 showed similar efficacy as Compound 1 as shown in Table 3 below.

Table 3. DA concentration (% of control) in striatum of MPTP-treated mice

Dosage Control MPTP only Compound 1 Compound 2 Compound 3 Compound 4 Dopamine (DA) concentration in striatum 20mg / kg, ip 100% 35.4% * 86.7% 144.9% 121.8% 172.5% Dopamine (DA) turnover rate 20mg / kg, ip 100% 46.9% * 103.0% 90.5% 85.4% 93.4%

The protective effect of Compound 1 against dopamine neuronal damage using the MPTP acute administration model showed that MPTP reduced the amount of dopamine in the striatum and that MPTP induced the damage of dopamine neurons to Compound 1,2,3 and 4. It was significantly inhibited by the cells and showed a brain cell protective effect. At this time, the recovery of dopamine content was reduced by MPTP. In the case of DOPAC, Compound 1 (20 mg / kg) was reduced in a concentration-dependent manner, which showed a significant MAO-B inhibitory effect. Compounds 2, 3 and 4 also showed similar efficacy in this model.

Therefore, the substituted benzene derivative compounds can be confirmed to have the effect of preventing or treating Parkinson's disease by showing the inhibitory activity of the dopamine neurons damage or brain cells.

3) TH immunohistochemical staining

In order to investigate the effects of MPTP damage on dopamine nervous system and drug-protective effects, TH (tyrosine hydroxyase) immunohistochemical staining was performed in striatum and medulla after 7 days of MPTP administration. When sectioned at a thickness of 40 μm in black matter sections from -2.06 mm to -4.04 mm based on Bregma, one sheet per six sections was taken out, and TH positive cells were continuously observed at high magnification (X200). Results After treatment with MPTP, TH positive cells in black matter decreased 35.5% (321.2 ± 7.0 TH + cells) in the MPTP group compared to the control group (488.5 ± 6.1 TH + cells). In comparison, 85.3% (416.7 ± 6.1 TH + cells) and 96.9% (473.3 ± 3.4 TH + cells) of TH-positive cells survived, indicating that Compound 1 significantly inhibited the reduction of TH-positive cells in the black matter by MPTP administration. Could be (FIGS. 3 and 4).

That is, as can be seen from Figures 3 and 4, in the group administered with Compound 1 significantly reduced neuronal cell damage in the group administered with Compound 1, while the number of DA neurons responding to TH was significantly reduced in the group administered with MPTP alone.

FIG. 3 shows TH immunoreactivity in black matter of control mice administered with saline only to mice administered with MPTP (3 × 20 mg / kg).

A. Control group B. MPTP monotherapy group C. Compound 1 20 mg / kg dose group D. Compound 1 40 mg / kg dose group

Data in FIG. 4 is mean ± SEM (n = 6).

* Is p <0.05 compared to control, # is p <0.05 compared to MPTP alone group.

A. Control group B. MPTP alone group C. MPTP + 1 compound (10 mg / kg) group D. MPTP + 1 compound (20 mg / kg) group

 The results for compounds 2, 3 and 4 showed a similar effect to compound 1 as shown in Table 4 below.

Table 4. On SNpc of MPTP-treated Mice

Control MPTP only Dosage Compound 1 Compound 2 Compound 3 Compound 4 100% 35.5% * 20mg / kg, ip 85.3% 71.0% * 96.4% 99.1%

 Number of TH positive neurons. (% of control)

Western blot analysis of the expression level of tyrosine hydrolase (TH) in substantia nigra (SN) showed a tendency to decrease TH expression in the MPTP group, whereas the expression level of the compound 1 group in the control group (sham-lesioned mice). Similar to Therefore, it was possible to confirm the protective effect of the nile neurons (FIG. 5).

In FIG. 5, the expression level of TH was decreased in the MPTP administration group, whereas the expression level of the compound 1 administration group was similar to that of the control group (sham-lesioned mice).

A. Control, B. MPTP alone, C. MPTP + Compound 1 (10 mg / kg), D.MPTP + Compound 1 (20 mg / kg), E. MPTP + Compound 1 (40 mg / kg)

Example  3: Parkinson's disease  6-animal model OHDA Rat  Effect on the model

6-OHDA (6-hydroxy dopamine), widely used as a Parkinson's disease-causing drug, is absorbed through the cell membranes of catecholaminergic neurons and stored and accumulated in monoamine storage granules to selectively store catecholaminergic neurons. Toxicity is present in cells (Ogawa et al., 1994). Animal models that destroy dopamine neurons in the unilateral brain with 6-OHD can be used as the control and can be used to compare spontaneous and drug-induced rotational movements (Perese et al., 1989). On the other hand, when 6-OHDA is injected directly into the striatum, 6-OHDA destroys the end of the dopamine nerve cell, which causes the cell body in the black matter to start to be destroyed from the 5th day after the 6-OHDA injection. Animal models can be produced that cause cell degeneration (Przedborski et al., 1995). Thus, the destruction of the dopamine nerve fibers in the striatum ultimately results in progressive degeneration of dopamine neurons in the black matter, similar to Parkinson's disease in humans compared to other models in which degeneration occurs rapidly. It can be used as an animal model suitable for observing variations in endogenous factors that appear.

Therefore, this experiment prepared 6 Parkinson's disease animal model showing gradual killing of dopamine neurons by injecting 6-OHD into striatum. Simultaneous administration with dopa was to evaluate the usefulness of Parkinson's disease.

1) Materials and Methods

end. Parkinson's disease animal model using 6-hydroxydopamine (6-OHDA)

8-week-old (205-300 g) male SD rats were anesthetized by intraperitoneal injection of 3 ml / kg of equithesin (Brundin et al., 1988). The anesthetized animal was fixed to 0 mm on a stereotaxic frame (David Kopf, USA) based on both interaural lines, and then the skull was perforated. 0.2 mg / ml ascorbic acid was used in the sham group, and 6-OHDA (10 ug / 2 ul free base in 0.2 mg / ml ascorbic acid) was used in the lesioned and drug treated groups. per minute using a Hamilton syringe (10 ul, 26G needles) in the forward (AP) -4.4 mm on the bregma (AP), side (ML) 1.2 mm and dura matter (DV) -8.0 mm) Inject at a rate of 0.4 ul. 6-OHDA was stored in ice in the shade before injection to prevent natural oxidation. The needle was left in place for 5 minutes after drug injection, the needle was removed at a rate of 1 mm per minute, and the incision site was closed.

I. Drug treatment and L-dopa-induced rotational motion measurement

To verify Parkinson's disease therapeutic effects of compounds 1, 2, 3 and 4, it was tested whether they could sustain the rotational movement induced by L-dopa. To this end, each compound was dissolved in 0.5% carboxymethylcellulose (CMC) and intraperitoneally administered at 20 mg / kg and rotational motion was measured for 30 minutes. In order to maintain the amount of levodopa in the brain after the rotational movement measurement, 12.5 mg / kg of benzrazide, an inhibitor of DOPA decarboxylase, was administered intraperitoneally in the periphery and levodopa (30 mg / kg) again 30 minutes later. Was administered intraperitoneally to induce rotational movement. Rotational motion was measured for a total of 2 hours at 5 minute intervals to compare net rotations per 5 minutes.

All. Effects on Apomorphine-induced Rotational Movement and L-dopa Rotational Responses: Net Rotational Counts

To confirm the disappearance of dopamine neurons, unilateral rotational movement was observed for 60 minutes by subcutaneous injection of apomorphine (apomorphine 0.5 mg / kg) in the back of the neck after 14 days after lesion induction. When dopamine neurons are killed and the concentration of dopamine in the striatum is reduced, the hypersensitivity of the dopamine receptors in the striatum is induced. It will rotate. Animals with at least 300 rotations were selected and treated to determine their efficacy. Rotational motion was measured using an automated rotometer as described by Ungerstedt (1970), where each rotational speed was measured as [net turns = contralateral rotation-ipsilateral Number of revolutions].

la. Statistical analysis

Five or more experiments were used to confirm the damage and protective effect of dopamine neurons following 6-OHDA injection and the experimental data were expressed as mean ± mean standard error (SEM). Statistical analysis was tested by Student's t-test after one-way ANOVA test, and was determined to be significant when p value was 0.05 or less.

2) Results: Behavioral effects of compounds 1, 2, 3 and 4 in 6-OHDA model

After 14 days after 6-OHDA injection, each compound was administered to measure unilateral rotational motion for 120 minutes. There was no change in unilateral net rotations with vehicle solution and significantly increased in the L-dopa only group. When intraperitoneally treated with L-dopa (8 mg / kg, intraperitoneal) and Compound 1 (20 mg / kg), the unilateral forward rotational speed was significantly increased compared to levodopa alone (n = 11). There was also a significant increase in the number of unilateral net rotations in the group administered 3, 4, or 4 simultaneously with levodopa (FIG. 6).

6 shows the result of measuring the net rotation rotated in the opposite direction that caused the lesion for 60 min. The number of experimental animals in each group was 11-14. All data are presented as mean ± SEM.

 # P <0.05 compared to control, ## P <0.05 compared to levodopa (L-dopa) alone group.

A. Control group B. L-dopa alone group (8 mg / kg, intraperitoneal administration) C. L-dopa + compound 1 (20 mg / kg, intraperitoneal administration) D. L-dopa + compound 2 administration group (20 mg / kg, Intraperitoneal administration) E. L-dopa + compound 3 administration group (20 mg / kg, intraperitoneal administration) F. L-dopa + compound 4 administration group (20 mg / kg, intraperitoneal administration)

In FIG. 6, it is considered that the longer the rotational motion is maintained in the group to which the representative compound 1,2,3 or 4 is administered compared to the group to which only levodopa is administered, they are effectively inhibiting MAO-B. Representative compounds 1, 2, 3 or 4 significantly increase the action time of levodopa, as the net rotational speed is significantly increased compared to the control group.

Example  4: Verification of therapeutic effect on depression

Animal models to validate depression treatment include forced swimming tests, tail suspension tests (TST), and 5-HTP-induced head twitch tests. Very well known.

Forcing mice to swim in a confined space, which is unavoidable in a forced swim test, is known to have an effect on depression, which can reduce the time of non-movement (Porsolt et al. , 1997,1998). Antidepressants, as well as stimulants such as amphetamines and caffeine, are well known to reduce inactivity time.

TST was developed as one of the animal models for evaluating antidepressants (Steru L. et al., (1985) Tail Suspension test: A new method for screening antidepressant in mice. Psychopharmacology 85; 367-370). The model was introduced on the basis that the immobile behavior seen from unavoidable stress (the tail is tied up and tied up) is similar to the depression in the human body (Steru L. et al). If you have a shortening effect of immobility time that does not show the behavior to escape, it can be said to have a treatment effect for depression.

Antidepressants have the function of enhancing the function of the noradrenergic / serotonergic system of the central nervous system. Some antidepressants work by blocking the resorption of serotonin. Based on this evidence, the administration of 5-hydroxytryptophan, a precursor of serotonin, with antidepressants causes head-twitch symptoms (Corne SJ et al., (1963). A method for assessing the effects of drugs on central actions of 5-hydroxytryptamine.Br. J. Pharmacol 20: 106-120]. Therefore, increased head-twitch symptoms have antidepressant effects.

Antidepressants with various mechanisms, as well as antidepressants, which are known to be closely associated with the maintenance of serotonin levels in the brain, are efficacious in this model.

1) Forced Swimming Test

end. Experiment method

In this experiment, ICR mice (20-25 g 3 weeks old) were prepared and then separated into 8 groups for each administration group, prepared and adjusted 1 hour before the experiment, and then prepared test compound 1 was administered (intraperitoneal / oral). After a certain period of time (30 minutes for intraperitoneal administration, 1 hour for oral administration) after administration to the control group and the experimental group, water maintained at a constant temperature at 25 ° C. was placed in a cylinder (a height of 25 cm and a diameter of 10 cm) up to 6 cm from the inlet. After filling, get the animals. The drug efficacy was evaluated by measuring the immobility time of the animal for 4 minutes after 2 minutes of adaptation time out of the total 6 minutes of measurement time.

I. Experiment result

Compound 1 was administered at 20 mg / kg dose intraperitoneally, and Compound 3 or 4 was orally administered at 60 mg / kg dose, respectively, and after 30 minutes of inactivity, the control time was compared to 100%. As a result, compounds 3 and 4 showed a significant reduction effect of 23.0% and 18.0 *% (p <0.05 vs. control group), respectively, and it was confirmed that they possess antidepressant effect.

2) Tail Suspension Test (TST)

end. Experiment method

ICR mice (20-25 g 3 weeks old) were prepared and then separated into eight groups for each administration group, prepared and adjusted 1 hour before the experiment, and then the prepared reagents were administered (intraperitoneal / oral). After a certain period of time (30 minutes for intraperitoneal administration, 1 hour for oral administration) after administering to 4 to 4, a round stainless rod of 1 cm in width is 16 cm wide, shielded by black wood left and right at 35 cm high. Experiment by fixing a cage of 40 cm. The efficacy was evaluated by measuring the animal's non-moving time for 4 minutes after 2 minutes of adaptation time out of a total of 6 minutes.

I. Experiment result

After 30 minutes of intraperitoneal administration of Compound 1 at a 30 mg / kg dose, the time of inactivity was measured and the control time of the control group was compared to 100%, indicating a significant difference of 26.3% * (p <0.05 vs. control group). One reduction effect was shown. Compound 2 showed a 20.5% reduction in oral administration at 60 mg / kg and Compound 4 had a 50% reduction in 43.0 mg / kg at oral administration. Compound 3 showed a 13.3% reduction at the 30 mg / kg dose upon intraperitoneal administration.

3) 5-HTP-induced hair twitch test

end. Experiment method

After preparing ICR mice (20-25g 3 weeks old), 8 dogs were separated for each administration group, prepared and adjusted 1 hour before the experiment, and then the prepared compound 1 was administered (intraperitoneal or oral). Carbidopa (25 mg / kg, intraperitoneal) is administered at 30 min after compound 1 is administered to the control and experimental groups, and 5-HTP (100 mg / kg) is administered at 45 min. The efficacy was evaluated by measuring the number of head cramps during the animal's behavior for 1 hour to 2 minutes after the administration of the reagent.

I. Experiment result

When the compounds 1,2,3 and 4 were administered orally at a dose of 30 mg / kg, the number of head cramps of the animals was increased to 100% of the control group for 1 hour to 2 minutes, and the result was 173.3%. .

Compound 2 showed 394.7%, 3 showed 200.0%, and 4 showed 420.0% increase.

Table 5. Effects on Depression

Compound number One 2 3 4 Forced Swimming Experiment (30mg / kg ip) 5.6% 2.4% at 60po 23.0% * at 60po 18.0% * at 60po Tail suspension experiment (30mg / kg ip) 26.3% * 20.5% at 60po 13.3% 43.0 po 5-HTP ^{1 )} Hair Cramp Experiment (30mg / kg po) 173.3% 394.7% * 200.0% 420.0% *

Compounds 1, 2, 3, and 4 exhibited antidepressant action in all three models, and thus, the substituted benzene derivative compounds of the present invention are considered to have antidepressant effects.

Example  5: Validation of epilepsy treatment effect

The maximum electric shock test is an animal model that causes general tonic-clonic epileptic seizures, and the strychinine-induced seizure test is an absence seizure or a myoclonic seizure animal. Well known as a model.

1) Max electric shock test

end. Experiment method

After preparing ICR mice (20 ~ 25g 3 weeks old), 8 dogs were separated for each administration group, prepared and adjusted 1 hour before the experiment, and the prepared reagents were administered (intraperitoneal administration / oral administration). Stabilizes the shock generator (IITC Inc) (90mA, 60Hz, 02sec)

After an appropriate time (0.5 hours, 1 hour, 2 hours, 4 hours) of administering the test substance to the experimental group, a shock generator (IITC inc, USA) was used to give an impact to the eye of the animal by applying a stimulus junction to the animal's eye. -Efficacy was evaluated by expression of -limb tonic extension.

I. Experiment result

Compound 1 was administered intraperitoneally at a dose of 5 mg / kg to 30 mg / kg, which significantly inhibited HL and showed a 50% inhibitory effect (ED ₅₀ ) of 18.6 mg / kg.

In addition, the same experiment orally showed significant efficacy and ED ₅₀ The value was 35.0 mg / kg. The efficacy for compounds 2, 3 and 4 is shown in Table 5 below.

2) Strychinine-Induced Seizure Testing

end. Experiment method

ICR mice (20-25 g 3 weeks old) were prepared and then separated into eight groups for each administration group, prepared and adjusted 1 hour before the experiment, and then the prepared compound 1 was administered (intraperitoneal administration / oral administration). Immediately before the experiment, strickin (100 mg / kg in saline) was prepared, and each group was treated with trikinin (100 mg / kg) subcutaneously after a titration time (0.5 hour, 1 hour, 2 hours, 4 hours) and for 0.5 hour. The efficacy of the animals was evaluated by the presence of Clonic Sezure for at least 5 seconds.

I. Experiment result

In the group administered Compound 1 at a dose of 30 mg / kg intraperitoneally, 100% suppression of hepatic seizures was observed.

Compound 2 had a 50% inhibitory effect (ED50) of 17.2 mg / kg upon intraperitoneal administration, Compound 3 had a 22.3 mg / kg upon intraperitoneal administration and Compound 4 showed an antiepileptic effect at a dose of 15.2 mg / kg (Table 6).

Table 6. Effects on Epilepsy

Compound number One 2 3 4 Electric shock (ED ₅₀ in mg / kg) 18.6ip 35.0po 24.7ip 62.6po 15.6ip 23.7po 15.9 ip 38.8po Strykinin-Induced Cramps (30mg / kg ip) 6/6, 2h 17.2ip, @ 0.5h 22.3ip, @ 2h 15.2ip, @ 2h

In these models, compounds 1,2,3 and 4 exhibited an effect of inhibiting seizures, and thus, the substituted benzene derivative compounds of the present invention are considered to have an epilepsy therapeutic effect.

Example  6: Effect confirmed in pain animal model

Pain occurs when irritating foreign bodies are administered into the abdominal cavity of mice. The animal then acts to stretch out the body, called writhing. This model is most commonly used to measure peripheral analgesic effects (Nakamura H. et al., (1986) Central and peripheral analgesic action of non-acidic non-steroidal anti-inflammatory drugs in mice and rats, Arch Int Pharmaco-dyn 282: 16-25].

Hot plate test is a pain measurement model based on the evidence that the feet of mice are very sensitive to heat that does not damage the skin. These include behaviors such as jumping, shrunk feet, and licking the feet. Painkillers that act on the central nervous system prolong the response time to these stimuli, but painkillers that act only on the peripheral nervous system generally No effect in the model (Kitchen I. et al., 1985) Assessment of hot-plate anti-nociceptive test in mice. A new method for the statistical treatment of graded data. J Pharmacol Meth 13: 1-7).

The formalin test was designed as a chronic pain animal model (Dubuisson D et al., (1977) The formalin test: A quantitative study of the analgesic effects of morphine, meperidine and brain stem stimulation in rats and cats.Pain 4: 161-174 )). In this method, the effects of drugs mainly on the central nervous system are confirmed, but the drugs on the peripheral side have little effect, and thus can be effectively used to distinguish between drugs that can act differently on inflammatory and non-inflammatory pain (Chau TT). (1989) Analgesic testing in animal models.In: Pharmacological Methods in the Control of Inflammation.Alan R. Liss, Inc., pp 195-212; Cowan A (1990) Recent approaches in the testing of analgesics in animals.In: Modern methods in Pharmacology, 6, Testing and Evaluation of Drug of Abuse.Wieley-Liss, Inc. pp 33-42)).

1) Writhing Test

end. Experiment method

ICR mice (27 ~ 30g 5 weeks old) were prepared and then separated into 8 groups for each experimental group and control group, prepared for 1 hour before the experiment, and then the prepared test substance was administered (ip / po). After a certain period of time (ip = 30 minutes, po = 1 hour) after administration to control and experimental groups, 0.8% acetic acid was injected intraperitoneally, and the efficacy was evaluated by the presence of torsional behavior seen in animals for 10 minutes. .

I. Experiment result

When Compound 1 was administered intraperitoneally at a dose of 10 mg / kg, the control group exhibited an inhibitory effect of 56.6% (p <0.05 vs. Control) when compared to 100% of the number of twists in the control group. Compound 2 showed 60.2% * (p <0.05 vs. Control), Compound 3 showed 58.7% * (p <0.05 vs. Control), and Compound 4 showed 61.7% * (p <0.05 vs. Control). 6).

2) Hot plate test

end. Experiment method

After maintaining a plate analgesia meter (Ugo Basile, Italy) at a constant temperature of 55 ℃, ICR mice (27-30g 5 weeks old) were prepared, and then separated into eight animals for each experiment group and control group 1 hour experiment Prepare and adapt and place the animal in a transparent cylinder 19 cm high and 17 cm high on a hot plate analgesic meter and measure the time for linking and hind legs tremble. After the preliminary test, the prepared test substance is administered. After a certain period of time (30 minutes for intraperitoneal administration, 1 hour for oral administration) after administration to the control group and the experimental group, the test was performed in the same manner as the preliminary test to measure the time for which forelimb linking and hind leg tremors appeared. The efficacy was assessed according to the preliminary test of the behavioral expression of the animals maintained at 50% or more than the time of the preliminary test and the change amount (% MPE) of the test.

I. Experiment result

The ED50 value of Compound 1 was 62.9 mg / kg for intraperitoneal administration and 72.7 mg / kg po for oral administration. Compound 2 was not as high as 4.3% intraperitoneally (30 mg / kg), ED5 of Compound 3 was 58.8 mg / kg (p <0.05 vs. Control) intraperitoneally and 93.9 mg / kg (p) <0.05 vs. Control) and Compound 4 was 20.2 mg / kg (p <0.05 vs. Control) for intraperitoneal administration and 57.7 mg / kg (p <0.05 vs. Control) for oral administration (Table 6).

3) Formalin test (late phase)

end. Experiment method

ICR mice (27-30g 5 weeks old) were prepared and then separated into 8 groups for each experimental group and control group prepared one hour before the experiment and adapted, and then administered the prepared test substance (ip / po). After a certain period of time (ip = 30 minutes, po = 1 hour) after administration to the control and experimental groups, 2 μl of 2.5% formalin was injected into the hind paw of the animal, and the animals were acclimated for 20 minutes, and then The effect of formalin-injected hind paws on the floor was measured and the efficacy was evaluated.

I. Experiment result

Compound 1 showed pain inhibition of 26.5% at 60 mg / kg upon intraperitoneal administration. Compound 2 showed 67.6% * (p <0.05 vs. Control), compound 3 showed 70.4% * (p <0.05 vs. Control), compound 4 showed 18.3% pain suppression effect (Table 7).

table. 7. Effects on Pain

Compound number One 2 3 4 Acetic acid distortion (10mg / kg ip) 56.6% * 60.2% * 58.7% * 61.7% * Thermal Plates (ED ₅₀ ) 62.9 ip 72.7 po 4.3% (30ip) 58.8 ip 93.9 po 20.2 ip 57.7 po Formalin (60mg / kg po)  26.5%  67.6% *  70.4% *  18.3%

Since compounds 1,2,3 and 4 all exhibited pain suppression in these pain animal models, it is believed that the substituted benzene derivative compounds of the present invention possess excellent therapeutic effects in acute and chronic pain.

Example  7: Effect confirmed in anxiety animal model

In addition to the typical defense instincts of animals, such as freezing, fleeing, and attacking, they are buried in a certain object that can be irritated with litter, showing the behavior of being freed from fear. burying) tests are generally well known animal testing methods used to verify anxiety effects (Njung'e K et al., 1991, Pharmacol Biochem Behav. 38: 63-67; Broekkamp CL et al., (1986), Eur. J. Pharmacol 126: 223-229); Broekkamp et al., 1986). Efficacy in this model, therefore, is evidence of a therapeutic effect on anxiety.

A) Marble Burying Test Method

ICR mice (27-30g 5 weeks old) were prepared and then separated into 8 groups for each experimental group and control group prepared one hour before the experiment and adapted, and then administered the prepared test substance (ip / po). After a certain period of time (ip = 30 minutes, po = 1 hour) after administration to the control group and the experimental group, put into a plastic cage containing 24 marbles and measure the efficacy by comparing the number of marbles buried for 30 minutes with the control group. It was.

B) Experimental results

Compound 1 showed an efficacy of 19.0 mg / kg ED50 when administered intraperitoneally. Compound 2 exhibited an anxiety effect with an ED 50 value of 17.4 mg / kg, Compound 3 18.7 mg / kg, and Compound 4 16.5 mg / kg (Table 8).

Table 8. Effects on Anxiety

compound One 2 3 4 Marbling in mice (MB) (ED ₅₀ in mg / kg ip) 19.0 17.4 18.7 16.5

Compounds 1,2,3 and 4 exhibited efficacy in this model, so the substituted benzene derivative compounds of the present invention are believed to possess anxiety therapeutic efficacy.

Example  8: effect on binding of receptors

The binding effects of the compounds 1,2,3 and 4 on Na + channel site 2, dopamine transporter, norepinephrine transporter, serotonin receptors (5-HT _2a and 5-HT _2c ) were confirmed (Table 9).

Table 9. Binding Effects on Channels, Transporters, and Receptors

Compound number 1% Suppression (at 1mM) 2% inhibition (at 1mM) 3% Inhibition (IC ₅₀ ) 4% inhibition (at 1mM) channel Sodium, site 2 9% 42% 31% 6.44mM Transport Dopamine 3% 19% 23% at 10 mM 8.25mM Norepinephrine 69% 74% 48% 0.61 mM Serotonin 5-HT _2A 34% 19% 7% 1.73mM 5-HT _2c 11% 19% 2% 5.06mM

The results are closely related to the neuroprotective effects possessed by the compounds 1,2,3 and 4 and the mechanism of action of inhibiting Parkinson's disease, epilepsy, pain, anxiety and depression.

* Reference Example:

IP: intraperitoneal administration

PO: oral administration

IC _5O: 50% inhibition concentration

ED ₅₀ : 50% Efficacy

MAO-A: Monoamine Oxidase A

MAO-B: Monoamine Oxidase B

Figure 1 shows the effect of compound 1 on the change of dopamine (DA) and its metabolite concentration in mice administered with MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine).

Figure 2 shows the effect of compound 1 on the dopamine turnover in the intrastriatal 7 days after MPTP administration.

Figure 3 shows the inhibitory effect of dopamine neuron damage of compound 1 in the black matter 7 days after MPTP administration.

Figure 4 shows the number of dopamine neurons immune response to tyrosine hydroxyase (TH) after 7 days in mice administered with MPTP.

Figure 5 shows the effect of compound 1 on the degree of tyrosine hydroxyase (TH) expression in the black matter 7 days after MPTP administration.

Figure 6 shows the change in net rotational movement induced by levodopa (L-dopa) and compounds 1, 2, 3 and 4 after 6-OHDA (6-hydroxy dopamine) in the striatum.

Claims (6)

A pharmaceutical composition for treating cranial nerve disease, comprising an effective amount of a substituted benzene derivative of formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier

Formula (I) Where R is selected from the group consisting of substituted and unsubstituted aryl and heteroaryl including benzene; Y ₁ and Y ₂ are independently selected from the group consisting of H and C ₁ to C ₃ alkyl; A is N, O, S, C-OR ', C = O, C (O) NH, NHC (O), CH ₂ , CH = CH, and OCH ₂ C (OR') H, where R 'is H Or a C ₁ to C ₅ substituted or unsubstituted alkyl group; B is selected from the group consisting of cyclic aromatic amines, guanidines and heterocyclic aliphatic amines; X is selected from the group consisting of H and halogen; m is an integer of 0 to 4; n is an integer of 0-5. The aryl of claim 1, wherein R is substituted with one or more substituents selected from the group consisting of F-, Cl- and Br-, halogen, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, -CF ₃ and -OCF ₃ And heteroaryl including thiazole, oxazole, imidazole, pyridine; Y ₁ and Y ₂ are independently selected from the group consisting of H, methyl, ethyl and isopropyl; A is selected from the group consisting of N, O, C═O, C—OH, NHC (O), CH ₂ , OCH ₂ CHOH and CH═CH; B is selected from the group consisting of imidazole, triazole, tetrazole, guanidine, pyrrolidine, piperidine and morphonyl; X is selected from the group consisting of H, F, Cl and Br; m is an integer from 0-2; n is a pharmaceutical composition for treating neurological disease, characterized in that the integer of 1 to 3. The method of claim 1, wherein the neurological disease is Parkinson's disease, epilepsy, depression, pain, anxiety and degenerative brain disease is a pharmaceutical composition for treating cranial nerve disease, characterized in that one of the neurological diseases selected from the group consisting of. Cerebral neurological diseases, including Parkinson's disease, epilepsy, depression, pain, anxiety and degenerative brain diseases, comprising administering to a mammal an effective amount of the pharmaceutical composition for treating cerebral neurological diseases according to claim 1. Treatment of 5. The brain disease according to claim 4, wherein the effective amount is administered in a unit dosage form containing 0.5 to 2 mg of the active ingredient of the composition of claim 1, in a total daily dose of 1 to 2 mg. Treatment method. The method of claim 5, wherein the daily dose is administered once or twice a day.

Images