KR20180038225A - Composition comprising diamine derivatives for preventing or treating stroke, damage of nerve cells, parkinson's disease or amyotrophic lateral sclerosis - Google Patents

Abstract

The present invention relates to a pharmaceutical composition comprising diamine derivatives, stereoisomers thereof or pharmaceutically acceptable salts thereof as active ingredients for preventing or treating stroke, nerve cell injury, Parkinson′s disease or Lou Gehrig′s disease, or to a food composition comprising diamine derivatives, stereoisomers thereof or pharmaceutically acceptable salts thereof as active ingredients for preventing or treating stroke, nerve cell injury, Parkinson′s disease or Lou Gehrig′s disease. The pharmaceutical composition or the food composition according to the present invention is effective for alleviating or preventing the stroke, nerve cell injury, Parkinson′s disease or Lou Gehrig′s disease.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing or treating stroke, nerve cell damage, Parkinson's disease or Lou Gehrig's disease, which comprises a diamine derivative, a composition for preventing or treating Parkinson's disease or Lou Gehrig's disease,

The present invention relates to a pharmaceutical composition or food composition for preventing or treating stroke, nerve cell damage, Parkinson's disease or Lou Gehrig's disease, which comprises a diamine derivative, a stereoisomer thereof or a pharmaceutically acceptable salt thereof as an active ingredient.

Adult mammals The central nervous system (CNS) axons of neurons are known to regenerate axons of the peripheral nervous system (PNS) rapidly while their ability to regenerate after injury is very limited. The limited regenerative capacity of CNS neurons is partly a characteristic of the CNS axons, but is also due to an unacceptable environment. The CNS mycelium is not the sole cause of the inhibitory function of neurite outgrowth, but it contains a number of inhibitory molecules that actively block axonal growth and thus constitute an important barrier to regeneration.

Stroke is a cerebrovascular disease that occurs when normal blood flow to the brain is compromised and the brain receives too much or too little blood, and functional recovery after stroke is associated with remodeling of the damaged brain, including cerebral angiogenesis and neurogenesis . Sudden obstruction of the cerebral artery leads to severe neurological impairment, while demyelination of the axon from multiple sclerosis causes gradual spread of neurological function and painful disturbances. Therefore, it is very important to induce axon formation and glial cell formation in cerebral apoplexy and to promote functional recovery after nerve injury.

Spinal cord injury is mainly caused by the compression of the spinal cord due to the displacement of the spine due to trauma. In addition to mechanical primary damage, immediate necrosis of the damaged cells leads to apoptosis which leads to apoptosis of neurons and white matter cells in the gray matter and demyelination of axons ), Which ultimately leads to permanent dysfunction. The pathophysiologic analysis showed that glutamate induced glutamate induced oxidative stress, ATP depletion, and anaerobic condition (ROS), which are initially liberated in the damaged area after spinal cord injury, ) And inflammatory mediators such as inflammatory mediators such as proinflammatory cytokines such as TNF-α and IL-1β and iNOS are known to be the main causes of apoptosis. In particular, the inflammatory response lasts not only in the early lesion but also in the long term. In particular, the inflammatory response caused by subcapsular apoptosis or axon degeneration in epidemics is a common pathologic feature in most neurological disorders, Its regulation is known to be important in preventing the spread of neurological diseases.

On the other hand, known methods of treating nerve growth factor in the treatment of neurological diseases include a gene therapy method in which stem cells are transplanted directly into stem cells or a therapeutic gene is delivered using a virus as a gene transfer vector. For example, in Korean Patent Publication No. 2007-0060721, a recombinant adenovirus is a replication defective viral vector in which the E1B region is deleted. Recently, as a therapeutic gene for the neurological diseases, Has been studied for achieving therapeutic effects on neuronal damage, and it has been disclosed that it is useful for prevention and treatment of nerve damage diseases.

However, despite the demonstration of efficacy in animal experiments, the gene therapy of neuronal damage that has been studied so far has not demonstrated a clinically significant therapeutic effect, and the gene transfer technology using viruses has a relatively high gene transfer efficiency And the clinical application is very difficult due to the safety problem of viral vectors. In addition, the gene therapy method has a disadvantage in that the repetitive production process of the substance is difficult and expensive. In addition, the existing non-viral gene therapy method is difficult to apply to the clinical application because of the problem of efficient gene transfer and transportation process of transferring the polymer-encapsulated polymer into cells.

Accordingly, the present invention provides a composition for preventing or treating stroke, neuronal cell damage, Parkinson's disease or Lou Gehrig's disease, which comprises a diamine derivative, in order to overcome the problem of treating neuronal damage or stroke by gene therapy.

The present invention provides a pharmaceutical composition for preventing or treating stroke, neuronal cell damage, Parkinson's disease or Lou Gehrig's disease, which comprises a diamine derivative, a stereoisomer thereof or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention also provides a food composition for preventing or ameliorating stroke, neuronal cell damage, Parkinson's disease or Lou Gehrig's disease, which comprises a diamine derivative, a stereoisomer thereof or a pharmaceutically acceptable salt thereof as an active ingredient.

The present inventors have completed the present invention by using diamine derivatives to prevent or treat stroke, nerve cell damage, Parkinson's disease or Lou Gehrig's disease.

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

Whenever a component is referred to as "comprising ", it is understood that it may include other components as well, without departing from the other components unless specifically stated otherwise.

As used herein, the term "prevention" means any action that inhibits or delays stroke, neuronal cell damage, Parkinson's disease or Lou Gehrig's disease by administration of the pharmaceutical composition. In addition, the above-mentioned "treatment" means any action that improves or alleviates the symptoms of stroke, nerve cell damage, Parkinson's disease or Lou Gehrig's disease by the administration of the pharmaceutical composition.

The stroke or cerebrovascular accident of the present invention is often referred to as a stroke, in which the blood vessels supplying blood to the brain are blocked or burst, resulting in death of brain cells in the injured area, resulting in loss of consciousness, speech disorders, It is a neurological symptom in which a disorder occurs. In the present specification, the stroke includes ischemic stroke (cerebral infarction) in which cerebral blood vessels are clogged and hemorrhagic stroke (cerebral hemorrhage) in which cerebrovascular firing occurs.

In the present invention, the nerve cell damage may mean traumatic brain injury or spinal cord injury.

The spinal cord injury (SCI) of the present invention includes traumatic spinal cord injury or non traumatic spinal cord injury. Such traumatic injuries include, but are not limited to, bending injury, vertical compression injury, hypersomnia injury, and flexion and rotation injury. The traumatic spinal cord injury may be due to traffic accidents, falls, sports injuries, assault, and the like. Such non-traumatic injuries include, but are not limited to, vascular dysfunction, arthritis, degenerative joint disease, spinal subluxation, myelitis, transverse sclerosis, spinal cord convulsion, or spinal tuberculosis.

The traumatic brain injury (TBI) of the present invention occurs when the brain is damaged by a sudden trauma. TBI can occur when the head strikes an object suddenly and violently, or when an object penetrates the skull and enters the brain tissue. Symptoms of TBI can be mild, moderate, or severe, depending on the degree of damage to the brain. Persons with a mild TBI may be able to maintain consciousness or experience loss of consciousness for a few seconds or a few minutes. Other symptoms of hardness TBI include headache, chaos, lightheadedness, dizziness, blurred vision or eye fatigue, echoes, bad taste in the mouth, fatigue or sleepiness, changes in sleep patterns, behavior Or mood changes, and memory, concentration, attention, or difficulty in thinking. People with moderate or severe TBI may have the same symptoms. In addition, it may also be associated with headache, repeated vomiting or nausea, convulsions or seizures, waking from sleep, enlargement of one or both pupils, obscure language, weakness or paralysis of limbs, loss of coordination and increased chaos, , Or nervousness.

Parkinson ' s disease of the present invention is characterized by the selective disappearance of dopaminergic neurons in the midbrain, resulting in dysfunction of the basal ganglia involved in motor functional tissues, resulting in a variety of exercise functions such as sustained tremors, stiffness, Of the disease.

Lou Gehrig's disease of the present invention, also called amyotrophic lateral sclerosis (ALS), is a disease accompanied by degeneration of both upper and lower motor neurons (UMN) and lower motor neurons (LMN).

The present invention provides a pharmaceutical composition for preventing or treating stroke, neuronal cell damage, Parkinson's disease or Lou Gehrig's disease comprising a diamine derivative represented by the following formula 1, a stereoisomer thereof or a pharmaceutically acceptable salt thereof as an active ingredient do.

[Chemical Formula 1]

In formula (1)

R ₁ and R ₂ are the same or different from each other and each independently hydrogen; -C (= O) - (C 1 -C 4 alkyl); Or a -C (= O) -O- (C 1 -C 4 alkyl).

In one embodiment of the present invention, at least one of R ₁ and R ₂ is hydrogen.

In another embodiment of the present invention, R ₁ and R ₂ are both hydrogen.

In one embodiment of the invention, one of R ₁ and R ₂ is hydrogen and the other is -C (= O) - (C ₁ -C ₄ alkyl); Or a -C (= O) -O- (C 1 -C 4 alkyl).

In another embodiment of the present invention, one of R ₁ and R ₂ is -C (═O) - (C ₁ -C ₄ alkyl) and the other is -C (═O) - (C ₁ - C ₄ alkyl).

In one embodiment of the present invention, the diamine derivative represented by Formula 1 is represented by Formula 2 or Formula 3 below.

(2)

(3)

In formulas (2) and (3)

R ₁ is hydrogen; -C (= O) - (C 1 -C 4 alkyl); Or -C (= O) -O- (C ₁ -C ₄ alkyl)

R ₃ and R ₄ are the same or different from each other and each independently C ₁ -C ₄ alkyl.

As used herein, "alkyl" can refer to straight or branched chain monovalent hydrocarbons, examples of which may include methyl, ethyl, n-propyl, i-propyl and butyl, Lt; / RTI >

In one embodiment of the present invention, the diamine derivative represented by the formula (1) is represented by any one of the following formulas (1-1) to (1-6).

In the present invention, stereoisomers of diamine derivatives may exist as optical isomers, racemates, racemic mixtures, single enantiomers, diastereomer mixtures and individual diastereomers. These isomers can be separated by conventional techniques, for example, by dividing the diamine derivative represented by the formula (1) by tube chromatography or HPLC. Alternatively, the stereoisomers of each of the diamine derivatives represented by formula (I) may be stereospecifically synthesized using optically pure starting materials and / or reagents of known sequence.

In the present invention, the pharmaceutically acceptable salt means a salt commonly used in the pharmaceutical industry. Examples of the salt include inorganic ion salts such as calcium, potassium, sodium and magnesium, hydrochloric acid, nitric acid, phosphoric acid, Inorganic acid salts prepared from iodic acid, perchloric acid, sulfuric acid and the like; But are not limited to, acetic, trifluoroacetic, citric, maleic, succinic, oxalic, benzoic, tartaric, fumaric, mandelic, propionic, lactic, glycolic, gluconic, galacturonic, glutamic, glutaric, Organic acid salts prepared from acids, ascorbic acid, carbonic acid, vanillyric acid, hydroiodic acid and the like; Methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and naphthalenesulfonic acid; Amino acid salts prepared with glycine, arginine, lysine and the like; And amine salts prepared by using trimethylamine, triethylamine, ammonia, pyridine, picoline, and the like. However, the types of salts used in the present invention are not limited by the listed salts.

Preferred salts for the present invention include hydrochloride, phosphate, sulphate, trifluoroacetate, citrate, bromate, maleate or tartrate, and preferred salts of the compounds of the present invention are those of formula 1-7 or 1-8 And the like.

[Chemical Formula 1-7]

[Chemical Formula 1-8]

In general, active oxygen is known to be one of the major causes of neuronal damage and regression. Active oxygen means oxygen that has been oxidized into the body during the breathing process and is produced in various metabolic processes, attacking living tissues and damaging cells. These reactive oxygen species irreversibly damage DNA, cellular constituent proteins and lipids in living cells. In particular, when a large amount of reactive oxygen species is produced in brain cells, the resulting oxidative stress may cause changes in intracellular mitochondrial structure and function, resulting in stroke, nerve cell damage, Parkinson's disease, Lou Gehrig's disease, and the like.

The diamine derivative of the present invention has an antioxidative effect and prevents the neuronal cell death by inhibiting oxidative stress which affects the nerve cell without treatment of another antioxidant to prevent and ameliorate nerve cell damage and neural degeneration And has an excellent effect.

Recently, it has been found that the inflammatory reaction is one of the main mechanism causing nerve damage and degeneration. Bovine glial cells, which are immune cells present in the central nervous system, can be activated by various extrinsic and endogenous substances. Activated small glial cells are activated by inflammatory cytokines such as TNF-α and IL-1β, nitrogen monoxide, prostaglandins, Produce, release. The production of these substances induces an immune response in the short term, but excessive production or sustained production induces the death of adjacent neurons, resulting in neuronal damage or neural degeneration. In addition, the substances released by the apoptotic neurons cause the activity of the apoptotic cells again, so that the damage or degeneration of the neuronal cells may be exacerbated.

Microglia are the cells that perform the primary immune function in the central nervous system. They maintain the shape of thin and long branches and thin cell bodies. When they enter from the outside or from inside, they are removed from these toxins To protect the nerve cells, it turns into an activated form with thick, short branches and fat cell bodies.

In contrast to normal microglial cells, the activated small glial cells stimulate proliferation, proliferate and induce cytokines such as TNF-α, IL-1β and IL-6, chemokines, inducible nitric oxide synthase ), COX-2 (cyclooxygenase-2), and the like to produce inflammatory mediators.

Activation of small glioblast cells protects nerve cells from exterminating bacteria or viruses, while removing damaged cells. Nitrogen produced by iNOS and prostaglandins, TNF-α, etc., produced by COX-2, As a result, the activation of small glial cells leads to deterioration of nerve cell damage.

FIG. 7 is a graph showing the activity of interleukin-1 beta (IL-1?), Interleukin-6 and tumor necrosis factor alpha (TNF-a), which are inflammatory factors. In the case of a composition comprising the diamine derivative represented by the formula (1), a stereoisomer thereof or a pharmaceutically acceptable salt thereof as an active ingredient according to the present invention, the effect of reducing the activity of inflammatory factors . Therefore, the diamine derivative represented by the formula (1) according to the present invention has an excellent effect for preventing and ameliorating nerve cell damage and neural degeneration.

Furthermore, the composition comprising the diamine derivative represented by the above formula (1), a stereoisomer thereof or a pharmaceutically acceptable salt thereof is used for the prevention or treatment of stroke, neuronal cell damage, Parkinson's disease or Lou Gehrig's disease, Is a disease that is associated with the death or damage of nerve cells.

For example, the loss of dopaminergic neurons in the substantia nigra is a cause of Parkinson's disease, and in patients suffering from neuronal damage, e.g., spinal cord injury, the killing of neurons is observed.

In recent years, a method of regenerating neurons by promoting the growth of neurites has been studied.

As described above, the composition comprising the diamine derivative represented by the formula (1), the stereoisomer thereof or the pharmaceutically acceptable salt thereof according to the present invention is capable of inhibiting damage or death of nerve cells using the antioxidative effect and anti- And at the same time, it promotes the growth of dendrites, thereby enhancing the expression of neurons.

Figs. 4 and 6 are diagrams showing the analysis of AO and BS of dendrites in the cerebral cortical layer II / III and the hippocampal CA1 region (CA1 of hippocampus), respectively. Examination of the results of the above figures confirms that the diamine derivative according to the present invention promotes the expression of water dendrites in vivo.

The pharmaceutical composition of the present invention does not increase the efficacy, but may further include components that are commonly used in pharmaceutical compositions and can improve odor, taste, and visual appearance. In addition, the pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable additive. Pharmaceutically acceptable additives include, for example, starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, sugar, arabic gum, pregelatinized starch, cornstarch, powdered cellulose, But are not limited to, hydroxypropylcellulose, opaques, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, white sugar, dextrose, sorbitol and talc. In addition, the pharmaceutical composition may include a substance which has been used alone or has a therapeutic or prophylactic activity for a stroke, neuronal cell damage, Parkinson's disease or Lou Gehrig's disease.

The pharmaceutical composition of the present invention comprises a pharmaceutically acceptable carrier and may be formulated for oral or parenteral administration for human or veterinary use. When the pharmaceutical composition of the present invention is formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants can be used. Solid form preparations for oral administration include tablets, pills, powders, granules and capsules, which may contain at least one excipient such as starch, calcium carbonate ( Calcium carbonate, Sucrose or Lactose, and Gelatin. In addition to simple excipients, lubricants such as magnesium, styrene, and talc may be used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions and syrups, and various excipients such as wetting agents, sweetening agents, 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 solutions, suspensions, emulsions, freeze-dried preparations and suppositories. Examples of non-aqueous solvents and suspensions include vegetable oils such as propylene glycol, polyethylene glycol and olive oil, injectable esters such as ethyl oleate, and the like. As a base for suppositories, witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like can be used.

The pharmaceutical composition of the present invention can be administered orally or parenterally in accordance with the intended method, and can be administered orally, parenterally or intraperitoneally, rectally, subcutaneously, intravenously, intramuscularly, It is preferable to select the injection method.

The pharmaceutical composition of the present invention may be administered to a subject to prevent or treat stroke, neuronal cell damage, Parkinson's disease or Lou Gehrig's disease. As used herein, the term "individual" refers to a disease caused by stroke, neuronal cell damage, Parkinson's disease or Lou Gehrig's disease, or a disease caused by a direct or indirect cause thereof, and administration of the pharmaceutical composition of the present invention Means mammals such as horses, sheep, pigs, goats, dogs and the like, including humans with a disease whose symptoms may be improved, preferably humans.

As used herein, the term "administering" means introducing a pharmaceutical composition of the invention into a subject by any suitable method. The route of administration may be oral or parenteral administration via any conventional route so long as it can reach the target tissue. In addition, the pharmaceutical composition of the present invention can be administered by any device that allows it to migrate to a target cell.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term, pharmaceutically effective amount, as used herein, means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dosage level will vary with the body weight, sex, age, , The activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and rate of release, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with another therapeutic agent, and may be administered sequentially or simultaneously with a conventional therapeutic agent. May be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without adverse effect, and can be easily determined by those skilled in the art.

The pharmaceutical composition of the present invention may further contain at least one active ingredient which exhibits the same or similar pharmaceutical activity besides the diamine derivative represented by the formula (1), a stereoisomer thereof or a pharmaceutically acceptable salt thereof.

The present invention also relates to a pharmaceutical composition comprising a therapeutically effective amount of a composition comprising a diamine derivative of formula (I), a stereoisomer thereof or a pharmaceutically acceptable salt thereof, for the treatment of stroke, neuronal cell damage, Parkinson's disease or Lou Gehrig's disease Prevention or treatment of cancer.

As used herein, the term "therapeutically effective amount" refers to a diamine derivative, its stereoisomer or its pharmaceutically acceptable salts, which is effective for the prevention or treatment of stroke, neuronal cell damage, Parkinson's disease or Lou Gehrig's disease Indicates the amount of salt possible.

The present invention also relates to a method for preventing or treating stroke, neuronal cell damage, Parkinson's disease, or Lou Gehrig's disease by administering to a mammal, including a human, a composition comprising the diamine derivative represented by Formula 1, a stereoisomer thereof or a pharmaceutically acceptable salt thereof Prevention or treatment of cancer.

The method for preventing or treating stroke, neuronal cell injury, Parkinson's disease or Lou Gehrig's disease comprises administering a composition comprising a diamine derivative represented by the above formula (1), a stereoisomer thereof or a pharmaceutically acceptable salt thereof, Not only does it deal with the disease itself, it also includes inhibiting or avoiding signs of it. In the management of disease, the prophylactic or therapeutic dose of a particular active ingredient will vary depending on the nature and severity of the disease or condition, and the route by which the active ingredient is to be administered. The frequency of dose and dose will vary with the age, weight and response of the individual patient. Appropriate dosing regimens can be readily selected by those of ordinary skill in the art which will of course consider these factors.

In addition, the method for preventing or treating stroke, nerve cell injury, Parkinson's disease or Lou Gehrig's disease of the present invention is a method for preventing or treating Parkinson's disease or Lou Gehrig's disease by administering a composition comprising the diamine derivative represented by the above formula (1), a stereoisomer thereof or a pharmaceutically acceptable salt thereof, A therapeutically effective amount of a further active agent that is therapeutically useful, wherein the additional active agent comprises a diamine derivative of Formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, And may exhibit synergistic or supplementary effects with the composition.

The present invention also relates to the use of a composition comprising a diamine derivative represented by the above formula (1), a stereoisomer thereof or a pharmaceutically acceptable salt thereof for the preparation of a medicament for the treatment of stroke, neuronal cell damage, Parkinson's disease or Lou Gehrig's disease . The diamine derivative represented by the above formula (1) for the preparation of a medicament may be mixed with an acceptable adjuvant, diluent, carrier or the like, and may be prepared as a combined preparation together with other active agents to have a synergistic action of the active ingredients.

The matters mentioned in the use, composition and treatment method of the present invention are applied equally unless they are mutually contradictory.

The present invention also relates to a food composition for preventing or ameliorating stroke, neuronal cell damage, Parkinson's disease or Lou Gehrig's disease comprising a diamine derivative represented by the following formula (1), a stereoisomer thereof or a pharmaceutically acceptable salt thereof as an active ingredient to provide.

[Chemical Formula 1]

In formula (1)

R ₁ and R ₂ are the same or different from each other and each independently hydrogen; -C (= O) - (C 1 -C 4 alkyl); Or a -C (= O) -O- (C 1 -C 4 alkyl).

The diamine derivative represented by the formula (1), a stereoisomer thereof or a pharmaceutically acceptable salt thereof may be all those described in the above-mentioned pharmaceutical compositions.

The present invention also provides a food composition comprising the diamine derivative represented by Formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof. The food composition may be added as it is or may be used in combination with other food or food ingredients, and may be suitably used according to conventional methods.

As used herein, the term "food composition" means food prepared and processed using raw materials or ingredients having useful functions in the human body.

There is no particular limitation on the kind of the food. Examples of the food which can be added to the composition comprising the diamine derivative represented by the formula (1), a stereoisomer thereof or a pharmaceutically acceptable salt thereof include various soups, beverages, tea, drinks, alcoholic beverages and vitamin complexes , And health food in a conventional sense. In addition to the above, the composition comprising the diamine derivative represented by the formula 1 of the present invention, a stereoisomer thereof or a pharmaceutically acceptable salt thereof may be used as various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, And salts thereof, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the food composition of the present invention may contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination.

The composition comprising the diamine derivative according to the present invention, its stereoisomer or its pharmaceutically acceptable salt as an active ingredient has an antioxidative effect and can inhibit the death of nerve cells due to oxidative stress affecting the nerve cell And is effective in preventing or treating stroke, nerve cell damage, Parkinson's disease or Lou Gehrig's disease by inhibiting the activity of an inflammatory factor.

In addition, the composition comprising the diamine derivative, the stereoisomer thereof or the pharmaceutically acceptable salt thereof according to the present invention as an active ingredient can promote the growth of the dendrites and enhance the expression of neurons, Damage, Parkinson's disease or Lou Gehrig's disease.

1 is a microscope (magnification: 40 magnification) of hepatic tissues of the non-treated group (WT-None), the vehicle administered group (5xFAD-vehicle) and the compound administered group (1-2x) .
Figure 2 shows AST (Aspartate Aminotransferase) and ALT (alanine aminotransferase) of the drug-treated group (WT-None), vehicle-administered group (5xFAD-vehicle) .
FIG. 3 is a graph obtained by treating the dendrites of the cerebral cortex II / III and the dendrites of the cerebral cortex II / III with a control (DMSO) and the formula (1-1) for 4 weeks and measuring them at an intensity of 100X using an optical microscope.
FIG. 4 is a diagram showing an analysis of AO and BS of dendrites of the cerebral cortex layer II / III.
FIG. 5 is a chart of the water dendrites in the hippocampal CA1 region (CA1 of hippocampus) treated with the control group (DMSO) and the formula (1-1) for 4 weeks and then measured with an optical microscope at 100X magnification.
FIG. 6 is a diagram showing analysis of AO and BS of dendrites in the hippocampal CA1 region (CA1 of hippocampus).
FIG. 7 is a graph showing the activity of interleukin-1 beta (IL-1?), Interleukin-6 and tumor necrosis factor alpha (TNF-a), which are inflammatory factors.

Hereinafter, the present invention will be described in detail by way of examples with reference to the drawings. However, the embodiments according to the present disclosure can be modified in various other forms, and the scope of the present specification is not construed as being limited to the embodiments described below. Embodiments of the present disclosure are provided to more fully describe the present disclosure to those of ordinary skill in the art.

Production example. Preparation of diamine derivatives

Production Example 1. Synthesis of Formula 1-1

Palladium / carbon catalyst was added to a solution of 4-nitroaniline dissolved in ethanol, and the mixture was stirred at reflux overnight. The reaction was filtered through celite and the compounds were separated using tube-chromatography. A white powder was obtained with a yield of about 95%.

Production example 2. Synthesis of formula 1-2

A solution of 4-nitroacetanilide in methanol was added palladium / carbon catalyst, and the mixture was refluxed for 8 hours and stirred. The reaction was filtered through celite and the compounds were separated using tube-chromatography. A brown powder was obtained with a yield of about 90%.

Example 1 Evaluation of antioxidative capacity

Trolox equivalent antioxidant capacity (TEAC) analysis was performed to evaluate the antioxidant capacity of tylenol structurally similar to the compounds of Formulas 1-1 and 1-1.

The antioxidative capacity of the compounds was tested by TEAC analysis in (a) ethanol or (b) M17 cell lysate.

(a) Blue ABTS cation radical to produce, ABTS (7.0 mM, ^(and ABTS ^+; Sigma; ABTS = ahjino 2,2'- bis (3-ethyl-benzothiazole-6-O sleepy acid) diammonium salt) Sigma) were dissolved in water (2.5 mM) in water and cultured in the dark at room temperature for 16 hours. The obtained ABTS cation radical was diluted in ethanol and the absorbance was measured at 734 nm. ABTS cation radical solution (200 L) was added to a 96-well plate and incubated at room temperature for 5 minutes on a plate reader. Compound or trolox (6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid dissolved in ethanol) was incubated with ABTS cation radical solution for 10 minutes at room temperature.

The inhibition rate (% inhibition = 100 x (A ₀ - A) / A ₀ ) was calculated as absorbance at 734 nm and plotted as a function of ligand concentration. The TEAC value of the compound was calculated as the ratio of the slope of the compound to the trolox, and the measurement was performed in triplicate.

(B) For antioxidant analysis using cell lysates, cells were seeded in 6-well plates and grown to about 80% to 90%. The cell lysate was modified in a conventional manner.

M17 cells were washed once with cold PBS (pH 7.4, GIBCO) and adherent cells were gently pipetted. The cell pellet was centrifuged (2,000 g at 4 ° C for 10 min) and the pellet was resuspended in 2 ml of ice cold assay buffer (5 mM potassium phosphate, pH 7.4; 0.9% sodium chloride (NaCl); 0.1% glucose) (5 seconds pulse, 3 times at intervals of 20 seconds between pulses). The cell lysate was centrifuged at 5,000 g for 10 minutes at 4 ° C.

The supernatant was removed and stored on ice until use. Standards and samples of 96-well plates were delivered by adding compound, metmyoglobin, ABTS and an appropriate amount of hydrogen peroxide for the supernatant (10 L) of cell lysate.

After incubation at room temperature for 5 minutes on a vibrator, absorbance was recorded at 750 nm. The concentration of antioxidant (% inhibition = 100 × (A ₀ - A) / A ₀ , A ₀ is the absorbance of the supernatant of cell lysate) was calculated as absorbance and performed in triplicate.

The results of the Trolox equivalent antioxidant capacity (TEAC) test as described above are shown in Table 1 below.

Trolox (1-1) Tylenol in Cell Lysates 1.00 1.11 ± 0.05 n.d.

In Table 1, n.d. Means not determined and may indicate that antioxidant capacity has not been measured. That is, it can be confirmed that the antioxidative capacity could not be measured in the structure of Tylenol similar to the present invention.

In addition, it can be confirmed that the diamine derivative of the present invention exhibits antioxidative effects similar to those of trolox. This may mean that nerve cell death can be suppressed by oxidative stress, which affects nerve cells, without treatment of other antioxidants.

Example  2. Assessment of brain absorption rate

Example  2-1. For Formulas 1-1, 1-2 and 1-6, Brain absorption rate  evaluation

In order to evaluate the brain absorption rate of the compounds, CD1 male rats (Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China)) were used, and the Association for Assessment and Accreditation of Laboratory Animal Care).

Compound (10 mg / kg; single dose; sterile water) was dosed by oral gavage. Blood was drained through 150 μl retro orbital puncture or cardiac puncture until 5 min and transferred to a coated K2 EDTA tube by spraying with an anticoagulant.

Blood samples were placed in ice and plasma samples (2,000 g, 5 min, 4 ° C) were obtained by centrifugation. Immediately after the blood was drawn, the mice were euthanized by CO ₂ inhalation and the whole brain was washed, washed with dry cold physiological saline on filter paper, collected and weighed. The brain samples were immediately homogenized with 3 times (v / w) homogenization solution (PBS).

Plasma samples and brain samples were supplemented with internal standards (propranolol) in acetonitrile (CH ₃ CN; protein precipitation). The mixture is vortexed for 2 minutes and centrifuged at 14,000 rpm for 5 minutes and the purified supernatant is used to determine the lowest limit of quantitation of the compound at 1 ng / mL (plasma) and 4 ng / mL (brain) lower limit of quantitation and LC-MS / MS (UPLC / MS-MS API-5500, Framingham, MA, USA). The supernatant was stored at -80 [deg.] C prior to analysis.

Table 2 shows the results of measurement of the absorption rate in the brain for Formulas 1-1, 1-2 and 1-6.

compound Matrix Concentration
(ng / mL or ng / g) Mean Standard Deviation
(SD) Coefficient of variation
(CV (%)) Individual (1-1) Plasma 1611 1051 2014 1559 484 31.0 Brain 1365 1487 2175 1676 437 26.1 CSF 1695 2708 5282 3228 1849 57.3 1-2 Plasma 86.0 22.2 351 153 174 113.9 Brain BQL BQL BQL BQL N / A N / A CSF 129 27.9 353 170 166 97.7 1-6 Plasma 3104 2925 2310 2780 416 15.0 Brain 212 153 161 175 32.2 18.4 CSF 876 563 556 665 183 27.5

Example  2-2. For the metabolites of Formulas 1-1, 1-2 and 1-6, brain Absorption rate evaluation

Table 3 shows the results of measurement of the metabolism of the metabolites of Formulas 1-1, 1-2 and 1-6 in the brain by the same method as in Example 2-1.

compound Metabolite Matrix Concentration
(ng / mL or ng / g) Mean Standard Deviation
(SD) Coefficient of variation
(CV (%)) Individual (1-1) 1-2 Plasma 1002 930 1297 1076 195 18.1 Brain 760 881 1123 921 185 20.1 CSF 824 1101 1479 1135 329 29.0 1-6 Plasma 5203 5994 5479 5559 401 7.22 Brain 721 890 711 774 101 13.0 CSF 1002 1669 1341 1337 334 24.9 1-2 (1-1) Plasma BQL BQL BQL BQL N / A N / A Brain BQL BQL BQL BQL N / A N / A CSF BQL BQL BQL BQL N / A N / A 1-6 Plasma 5980 4639 5955 5525 767 13.9 Brain 1275 487 1230 997 443 44.4 CSF 2725 1045 2578 2116 930 44.0 1-6 (1-1) Plasma BQL BQL BQL BQL N / A N / A Brain BQL BQL BQL BQL N / A N / A CSF BQL BQL BQL BQL N / A N / A 1-2 Plasma 17.5 BQL 8.64 13.1 N / A N / A Brain BQL BQL BQL BQL N / A N / A CSF BQL BQL BQL BQL N / A N / A

BQL in Tables 2 and 3 means below the quantifiable limit, and N / A means not available.

The results of Table 3 show that the diamine derivative represented by Formula 1-1 of the present invention changes into a metabolite of the diamine derivative represented by Formulas 1-2 and 1-6 in vivo after administration, And the diamine derivative represented by the general formula 1-2 is changed into a metabolite of the diamine derivative represented by the general formula 1-6 in vivo after the administration and is absorbed into the brain or spinal fluid. Therefore, it can be confirmed that the diamine derivative of the present invention is absorbed into the brain or cerebrospinal fluid as a diamine derivative itself or a metabolite of a diamine derivative.

That is, as a result of Tables 2 and 3, it can be confirmed that the diamine derivative according to the present invention is smoothly absorbed into brain or CSF (Cerebrospinal Fulid). This means that the diamine derivative according to the present invention can be easily absorbed into the brain or spinal fluid, so that the pharmaceutical composition or food composition according to the present invention can easily reach the nerve cell, and thus can prevent stroke, neuronal cell damage, Parkinson's disease or Lou Gehrig's disease Or can be suitably used for treatment.

Example  3. Single dose toxicity measurement

Single-dose toxicity to the formula (1-1) and the formula (1-2) was measured in SD rats, 10 rats, 10 males and 5 females and 5 males for 1 day (25, 50, 100, 200, 400 mg) was directly administered to the stomach using a syring tube equipped with a sonde for once oral administration, (LD ₅₀ ) obtained by oral administration with dilution of DMSO / PEC 400 / DW = 1/5/4 mg once daily for 15 days and measuring the half-life (LD ₅₀ ) are shown in Table 4 below.

compound Lethal dose (LD ₅₀ : mg / kg) (1-1) 80 mg / kg 1-2 > 2,000 mg / kg

As a result of the above half-life lethal dose, the diamine derivative of the present invention has low toxicity and the composition comprising the diamine derivative, the stereoisomer thereof or the pharmaceutically acceptable salt thereof as an active ingredient can be used for the treatment of stroke, neuronal cell damage, Parkinson's disease or Lou Gehrig's disease It is confirmed that the present invention is suitably applied as a pharmaceutical composition or food composition for prevention or treatment.

Example  4. Hepatotoxicity  Experiment ( Hepatotoxicity  Studies)

Through the hepatotoxicity test when the diamine derivative of Formula 1-2 of the present invention was injected into the peritoneal cavity of 5x FAD animal model mice for a long period of one month or more (35 days) at 2 mg / kg every day, the hepatotoxicity of the compound of the present invention Respectively.

5xFAD animal model after administration of formula 1-2 In addition to the beneficial therapeutic efficacy of brain in brain, hepatotoxicity is a typical evaluation method for the expression of hepatotoxicity, hepatocellular pathology, blood aspartate aminotransferase (ALT) and alanine aminotransferase Change.

FIG. 1 is a graph showing the results obtained by injecting 2 mg / kg of compound of formula 1-2 (5xFAD-1-2) into peritoneal cavity of 5xFAD mice for 35 days, fixing liver with paraformaldehyde fixative, The sections were cut into 20 νm sections and stained with hematoxylin and Eosin (H & E).

1, the liver of the 5xFAD-vehicle-treated group (5xFAD-vehicle) of the control group had no morphologic difference compared to that of the non-drug-treated group WT-None mice, Or accumulation did not appear to cause morphological damage to the liver.

Likewise, no changes were observed in the 5xFAD mice (5xFAD-vehicle) or the drug-untreated mice group (WT-None) in the group administered with the compound of the formula 1-2 (5xFAD-1-2). Hepatocellular necrosis as well as portal tracts, fibrosis, or inflammation were not observed in any of the groups compared to the control group.

In order to investigate the effect of the diamine derivatives of formula (1-2) according to the present invention on the liver function in the dementia model mice, a representative hepatic function test, Aspartate Aminotransferase (AST), alanine amino group transfer Enzyme (alanine aminotransferase, ALT) content test was performed.

Figure 2 shows AST (Aspartate Aminotransferase) and ALT (alanine aminotransferase) of the drug-treated group (WT-None), vehicle-administered group (5xFAD-vehicle) .

Blood was collected from the portal vein immediately after killing the mouse (5xFAD-1-2) administered with the compounds of the formula 1-2 as used in the various tests, and the aspartate aminotransferase (AST), the alanine amino transferase Aminotransferase, ALT) were analyzed biochemically. As a result, no significant change was observed in all the experimental groups (WT-None, 5xFAD-vehicle, 5xFAD-1-2).

1 and 2, it was confirmed that the compounds of the present invention did not cause any hepatotoxicity or hepatic dysfunction when the mice were intraperitoneally injected at a dose of 2 mg / kg for 30 days or more.

Example  5. Experiments on the enhancement of neural cell expression

(1) Animal and drug administration

Wild-type C57BL / 6J rats were purchased from Hyochang Science and maintained according to the protocol approved by UNIST's Animal Welfare and Use Committee. To conduct experiments on neuronal spinogenesis, mice were injected with control (DMSO), Formula 1-1 (1 mg / kg) daily for 4 weeks.

(2) Morphological analysis of Golgi staining and dendrites

The FD Rapid GolgiStain Kit (FD NeuroTechnologies, Ellicott City, MD, USA) was used to analyze the dendritic densities and morphology of the brain. The dissected rat brain was immersed in solution A and solution B in a dark room at room temperature for 2 weeks, transferred to solution C, and placed at 4 ° C for 24 hours. The brain was sliced to a thickness of 100 μm using Microm HM560 (Thermo Scientific, USA). Dendritic images were acquired using a BX53 (Olympus, Japan) optical microscope. The density of the Cortical Layers II / III and CA1 of the hippocampus was measured using Cellsens software (Olympus, Japan). Images were coded, and dendrites were calculated in a blinded fashion. The dendrite was included in the analysis for a length of 0.2 to 2 μm. All morphological analyzes were done in a blinded fashion.

(3) Experimental results

The dendritic complexity of the dendritic complexes in the body of the compound of formula (1-1) was examined. Wild-type rats were injected with control (DMSO) and Formula 1-1 (1 mg / kg) for 4 weeks. After 4 weeks, the apical oblique (AO) and basal shaft (BS) of dendritic density in the cerebral cortex layer II / III and CA1 region of the hippocampus (CA1 of hippocampus) were analyzed.

FIG. 3 is a graph obtained by treating the dendrites of the cerebral cortex II / III and the dendrites of the cerebral cortex II / III with a control (DMSO) and the formula (1-1) for 4 weeks and measuring them at an intensity of 100X using an optical microscope.

FIG. 5 is a chart of the water dendrites in the hippocampal CA1 region (CA1 of hippocampus) treated with the control group (DMSO) and the formula (1-1) for 4 weeks and then measured with an optical microscope at 100X magnification.

FIG. 4 is a diagram showing an analysis of AO and BS of dendrites of the cerebral cortex layer II / III.

FIG. 6 is a diagram showing analysis of AO and BS of dendrites in the hippocampal CA1 region (CA1 of hippocampus).

Analysis of Figs. 4 and 6 showed that the rats treated with formula (1-1) had dendritic density ((n = 14; by 17%, * P < 0.05) in the cerebral cortical layer II / (n = 14; by 37%, ** P < 0.001). As a result, it was confirmed that the diamine derivative according to the present invention promotes the expression of dendrites in vivo. In other words, the promotion of the expression of the dendritic lobes as described above may mean that the expression of neurons is promoted. In this case, it may be expected that the neuronal damage or neural degeneration can be prevented and improved.

Example  6. Anti-inflammatory  Ability experiment

Inflammatory cytokines (IL-1β, IL-6 and TNF-α) for the anti-inflammatory effect of compounds of formula (1-1) and B1-Ac in macrophages cell lines (RAW264.7 cells) Respectively.

Enzyme-Linked Immunosorbent Assay (ELISA) was performed to measure the amount of cytokines in the cell culture medium. Cells were treated with 1M, 10M, 20M, and 50M, respectively, and then treated with 100 ng / mL of LPS. After 12 hours, cell cultures were collected for cytokines, IL-1β (DY401, R & D systems), IL-6 (DY406, R & D systems) and TNF-α (DY410, R & D systems).

50 μl of the culture was added to 96 well plates (32296, SPL Life Sciences) coated with capture antibodies of cytokines, and reacted at room temperature for 3 hours.

After washing three times with washing buffer, 100 μl of biotinylated antibody reagent was added to each well, reacted at room temperature for 2 hours, washed three times, and 100 μl of streptavidine-HRP solution was added to each well, incubated at room temperature for 20 minutes, and then washed three times with washing buffer. 100 μl of di (2-ehtylhexyl) -2,4,5-trimethoxybenzalmalonate (TMB) susbtrate (00-4201, eBioscience) was reacted for 5-30 minutes and then 50 μl of stop solution was treated with ELISA reader Absorbance was measured at 450 nm.

FIG. 7 is a graph showing the activity of interleukin-1 beta (IL-1?), Interleukin-6 and tumor necrosis factor alpha (TNF-a), which are inflammatory factors.

From the results of FIG. 7, it can be confirmed that the activity of the inflammatory factor is reduced when the diamine derivative of the present invention is treated. As a result, in the case of a composition comprising the diamine derivative represented by the formula 1 according to the present invention, a stereoisomer thereof or a pharmaceutically acceptable salt thereof as an active ingredient, It can be confirmed that it is effective in preventing and improving degeneration.

Claims (9)

A pharmaceutical composition for preventing or treating stroke, neuronal cell damage, Parkinson's disease or Lou Gehrig's disease comprising a diamine derivative represented by the following formula 1, a stereoisomer thereof or a pharmaceutically acceptable salt thereof as an active ingredient:
[Chemical Formula 1]

In formula (1)
R ₁ and R ₂ are the same or different from each other, and each independently hydrogen; -C (= O) - (C 1 -C 4 alkyl); Or a -C (= O) -O- (C 1 -C 4 alkyl). The method according to claim 1,
Wherein at least one of R &lt; ₁ &gt; and R &lt; ₂ &gt; is hydrogen, a stereoisomer thereof or a pharmaceutically acceptable salt thereof as an active ingredient for preventing or treating Parkinson's disease or Lou Gehrig's disease A pharmaceutical composition. The method according to claim 1,
The diamine derivative represented by the general formula (1) is represented by the following general formula (2) or (3), a stroke, neuronal cell damage, Parkinson's disease or Lou Gehrig's disease comprising the diamine derivative, its stereoisomer or a pharmaceutically acceptable salt thereof as an active ingredient A pharmaceutical composition for preventing or treating diseases,
(2)

(3)

In formulas (2) and (3)
R ₁ is hydrogen; -C (= O) - (C 1 -C 4 alkyl); Or -C (= O) -O- (C ₁ -C ₄ alkyl)
R ₃ and R ₄ are the same or different from each other, and each independently is C ₁ -C ₄ alkyl. The method according to claim 1,
The diamine derivative represented by the general formula (1) is represented by any one of the following general formulas (1-1) to (1-6), a stroke comprising the stereoisomer thereof or a pharmaceutically acceptable salt thereof as an active ingredient, , Parkinson's disease or Lou Gehrig's disease.

The method according to claim 1,
Wherein the pharmaceutically acceptable salt of said diamine derivative is a hydrochloride salt, a pharmaceutical composition for preventing or treating stroke, nerve cell damage, Parkinson's disease or Lou Gehrig's disease. The method according to claim 1,
Wherein said nerve injury is traumatic brain injury or spinal cord injury, neuronal cell damage, Parkinson's disease or Lou Gehrig's disease. A food composition for preventing or ameliorating stroke, neuronal cell damage, Parkinson's disease or Lou Gehrig's disease comprising a diamine derivative represented by the following formula (1), a stereoisomer thereof or a pharmaceutically acceptable salt thereof as an active ingredient:
[Chemical Formula 1]

In formula (1)
R ₁ and R ₂ are the same or different from each other, and each independently hydrogen; -C (= O) - (C 1 -C 4 alkyl); Or a -C (= O) -O- (C 1 -C 4 alkyl). 8. The method of claim 7,
The diamine derivative represented by the general formula (1) is represented by the following general formula (2) or (3), a stroke, neuronal cell damage, Parkinson's disease or Lou Gehrig's disease comprising the diamine derivative, its stereoisomer or a pharmaceutically acceptable salt thereof as an active ingredient Food composition for preventing or improving disease:
(2)

(3)

In formulas (2) and (3)
R ₁ is hydrogen; -C (= O) - (C 1 -C 4 alkyl); Or -C (= O) -O- (C ₁ -C ₄ alkyl)
R ₃ and R ₄ are the same or different from each other, and each independently is C ₁ -C ₄ alkyl. 8. The method of claim 7,
The diamine derivative represented by the general formula (1) is represented by any one of the following general formulas (1-1) to (1-6), a stroke comprising the stereoisomer thereof or a pharmaceutically acceptable salt thereof as an active ingredient, , Parkinson's disease or Lou Gehrig's disease.

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