KR20210134552A - Pharmaceutical composition for inhibiting inflammation of spinal cord injury or spinal stenosis - Google Patents

Abstract

The present invention relates to a pharmaceutical composition comprising 3-(4-chlorophenyl)-4-(4-aminosulfonyl-phenyl)-1-methyl-1H-pyrrole-2,5-dione or a pharmaceutically acceptable salt thereof for preventing or treating spinal cord injury and spinal stenosis. As the present inventors newly found that a compound according to the present invention inhibits activities of pro-inflammatory cytokines and PGE_2, which are inflammation mediators, to suppress the inflammation or pain of spinal cord injury or spinal stenosis, the compound according to the present invention can effectively suppress the inflammation or pain attributed to spinal cord injury or spinal stenosis and as such, is expected to be advantageously used in preventing or treating spinal injury or stenosis.

Description

Pharmaceutical composition for inhibiting inflammation of spinal cord injury or spinal stenosis {PHARMACEUTICAL COMPOSITION FOR INHIBITING INFLAMMATION OF SPINAL CORD INJURY OR SPINAL STENOSIS}

The present invention relates to a spinal cord comprising 3-(4-chlorophenyl)-4-(4-aminosulfonyl-phenyl)-1-methyl- 1H -pyrrole-2,5-dione or a pharmaceutically acceptable salt thereof It relates to a pharmaceutical composition for preventing or treating injury or spinal stenosis, and more particularly, to a composition for preventing or treating spinal cord injury or stenosis by inhibiting an inflammatory mediator of the spinal cord injury or spinal stenosis.

At the same time, the present invention relates to a pharmaceutical composition for inhibiting inflammation of a spinal cord injury or spinal stenosis.

Spinal stenosis refers to a medical condition in which the spinal canal surrounded by soft tissues constituting bones and nerve structures is narrowed, and causes intermittent claudication, pain in the lower extremities, and inability to walk.

Surgical decompression is the most commonly suggested surgical treatment option for the treatment of spinal stenosis, but it is difficult to predict the success of the operation, and it can cause muscle atrophy by destroying anatomical support structures such as myofibrillar ligaments due to laminectomy. Because of the risk, a method of treating spinal canal stenosis by applying a drug targeting a biomaterial involved in inflammation or pain is preferred.

Drugs for spinal stenosis mainly include anti-inflammatory drugs, analgesics, muscle relaxants, and steroid injections. Among them, steroids are adrenal corticosteroids with immunosuppressive and strong anti-inflammatory effects, and are widely used as a treatment for central nervous system demyelinating diseases such as multiple sclerosis. , weight gain, weakened immunity and increased risk of infection, peptic ulcer, myopathy, bone necrosis, cataracts, skin changes, and behavioral disorders.

Spinal cord injury refers to an injury that causes a temporary or permanent change in the function of the spinal cord, and causes loss of sensation in the body part located below the injured part, loss of muscle function, and pain in the lumbar spine.

Treatment of spinal cord injury is largely divided into drug treatment and surgical treatment. In the case of drug treatment, it has been reported that administering a large amount of steroids for acute spinal cord injury is effective in recovery, but this also includes the use of steroids for the stenosis. It has the same side effects as side effects, so there is a disadvantage in use.

Accordingly, as anti-inflammatory agents for the treatment of spinal cord injury or spinal stenosis, coxib drugs such as celecoxib and rofecoxib may be considered, but in 2004, such as Merck's rofecoxib, Coxib drugs have been banned because of their excessively increased risk of heart attack and stroke with long-term use.

In addition, as it has been confirmed in some studies that the adverse effects of rofecoxib on the cardiovascular system may be intrinsic chemical properties related to the metabolism of rofecoxib, the discovery of a new compound structure is urgently required. Studies on the subject are also being actively conducted (British Journal of Pharmacology (2012) 165, Shin et al.).

As a result of research to discover compounds effective for spinal cord injury or spinal stenosis, the present inventors confirmed that the composition containing the compound of the present invention inhibits the expression of pro-inflammatory mediators that cause inflammation or pain, the present invention was completed.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating symptoms caused by spinal cord injury or spinal stenosis, comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[Formula Ⅰ]

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention as described above, the present invention

Provided is a pharmaceutical composition for preventing or treating symptoms caused by spinal cord injury or spinal stenosis, comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[Formula Ⅰ]

In addition, the present invention provides a pharmaceutical composition, characterized in that the spinal stenosis is lumbar spinal stenosis (Lumbar spinal stenosis).

In addition, the present invention provides a pharmaceutical composition, characterized in that the composition inhibits intermittent claudication, paralysis, sensory loss, paresthesia, sensory impairment, inflammation or pain.

In addition, the present invention provides a method for preventing or treating symptoms due to spinal cord injury or spinal stenosis, comprising administering the composition to a subject.

In addition, the present invention provides the use of the composition for preventing or treating symptoms due to spinal cord injury or spinal stenosis.

The present inventors provide a compound comprising 3-(4-chlorophenyl)-4-(4-aminosulfonyl-phenyl)-1-methyl- 1H -pyrrole-2,5-dione or a pharmaceutically acceptable salt thereof It has been newly discovered that it inhibits inflammation or pain of spinal cord injury and spinal stenosis. The compound according to the present invention can effectively inhibit inflammation or pain caused by spinal cord injury or spinal stenosis, so that the prevention or treatment of the injury or stenosis is It is expected that it will be useful in making it happen.

Fig. 1 relates to the production of an animal model for inducing chronic mechanical allodynia through cauda equina compression. Fig. 1a shows a target lumbar region, Fig. 1b shows a silicone block used for manufacturing the animal model, and Fig. 1c shows a silicon block inserted into the target lumbar region.
2 shows the inhibitory effect _{of LPS-induced PEG 2} production of the compound according to the present invention.
Figure 3 relates to the results of confirming the infiltration of macrophages in the cauda equina of rats induced by chronic mechanical allodynia, Figure 3a is the results of the rotarod test of the chronic mechanical allodynia-induced animal model and the mock control group (false surgery group) 3b shows the results of measuring the paw withdrawal threshold (PWT) of the chronic mechanical allodynia-induced animal model and the simulated control group (sham surgery group), and FIG. 3c is the chronic mechanical allodynia-induced animal model. The results of observation of ED-1 positive macrophages in the compressed and non-compressed regions after pressing the cauda equina are shown.
Figure 4 relates to the results of confirming the role of the inhibitor in mechanical pain and inflammation induced by cauda equina pressure, and Figure 4a shows the PWT measurement results when celecoxib is applied to a chronic mechanical allodynia-induced animal model. 4b shows the PWT measurement results when the compound according to the present invention is applied to an animal model inducing chronic mechanical allodynia, and FIG. 4c is when celecoxib and the compound according to the present invention are applied to an animal model inducing chronic mechanical allodynia shows the expression results of TNF-α, interleukin-1β (interleukin-1β, IL-1β), IL-6, inducible nitric oxide synthase (iNOS) mRNA after 30 minutes, and FIG. 4d shows celecoxib and the present invention. It shows the results of measuring the relative expression levels of the inflammatory mediators when the compound according to the present invention was applied to the chronic mechanical allodynia-induced animal model, and FIG. Shows the results of measuring the expression level of PEG _{2 in the case.}

Hereinafter, the present invention will be described in detail.

The present invention relates to a pharmaceutical composition for treating or preventing spinal cord injury or spinal stenosis, comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[Formula Ⅰ]

The compounds of the present invention are 1- H pyrrole or derivatives of furan-2,5-dione, 3-(4-chlorophenyl)-4-(4-aminosulfonyl-phenyl)-1-methyl- 1H -pyrrole It is called -2,5-dione.

As used herein, the term "prevention" refers to any action that suppresses or delays the onset of spinal cord injury or spinal stenosis by administration of the pharmaceutical composition according to the present invention.

As used herein, the term "treatment" refers to any action in which the symptoms of spinal cord injury or spinal stenosis are improved or beneficially changed by administration of the pharmaceutical composition according to the present invention.

As used herein, the term "salt" is useful as an acid addition salt formed by a pharmaceutically acceptable free acid. Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, and aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanes. It is obtained from non-toxic organic acids such as dioates, aromatic acids, aliphatic and aromatic sulfonic acids. Such pharmaceutically non-toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, ioda. Id, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate , sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methylbenzoate Toxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, beta-hydroxybutyrate, glycerol cholate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate or mandelate.

The acid addition salt according to the invention is prepared by a conventional method, for example, by dissolving the compound in an aqueous solution of an excess of acid, and precipitating the salt using a water-miscible organic solvent, for example methanol, ethanol, acetone or acetonitrile. It can be manufactured by It can also be prepared by evaporating the solvent or excess acid from the mixture and drying the mixture, or by suction filtration of the precipitated salt.

In addition, a pharmaceutically acceptable metal salt may be prepared using a base. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and evaporating and drying the filtrate. In this case, it is pharmaceutically suitable to prepare a sodium, potassium or calcium salt as the metal salt. The corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable negative salt (eg silver nitrate).

In addition, the compound of the present invention includes all salts, isomers, hydrates and solvates that can be prepared by conventional methods as well as pharmaceutically acceptable salts.

The disease targeted by the present invention, "spinal cord injury (SCI)" is a spinal cord injury as the spinal cord (central nerve existing in the spine) is damaged by an accident or disease, or the spinal cord is damaged by a disease Symptoms include, but are not limited to, movement and sensory paralysis caused by the failure of nerve transmission between the brain and the body to occur properly.

In addition, the spinal canal is a tube-shaped hollow in the middle of the spine, and the intervertebral foramen is created by the lower and upper vertebrae, and the middle tube becomes a passageway through which nerves (spinal cord) pass from the brain to the limbs. The tube shape is oval or triangular, the largest in the cervical vertebrae (neck side), narrows in the thoracic vertebrae (chest side), grows again in the lumbar vertebrae (waist side), and narrows toward the bottom. stenosis)" is a disease in which the central spinal canal, nerve root canal, or intervertebral foramen narrows, causing pain in the lower back or complex neurological symptoms in the legs. Spinal stenosis mainly occurs in the lumbar region, and in general, spinal stenosis refers to lumbar spinal stenosis. it means to

In addition, "lumbar spinal stenosis (LSS)" refers to a disease in which the spinal canal surrounded by the soft tissues constituting the bones and nerve structures of the lumbar spine is narrowed. Causes of the spinal canal stenosis include lumbar spondylolisthesis, slipped disk, ligamentous thickening, and spinal degeneration due to aging. Symptoms of the spinal canal stenosis according to the present invention include intermittent claudication, pain in the lower extremities, inability to walk, compression of cauda equina nerve fibers, hypersensitivity, sensitization of the central nervous system and peripheral nervous system ( sensitization), which may include, but is not limited to, severe neuropathic pain, paralysis, hypoesthesia, paresthesia, and sensory impairment.

Meanwhile, the neuroinflammatory response is known to play an important role in the development and maintenance of spinal neuropathy pain. After tissue damage, immune cells migrate to the site of damage to produce pro-inflammatory cytokines and mediate the inflammatory response. Pro-inflammatory cytokines include TNF-α, IL-1β, IL-6, iNOS, and prostaglandin E2, which sensitizes neuronal pain transmission.

In one embodiment of the present invention, the compound of the present invention effectively inhibits the pain of spinal stenosis by administering the compound of the present invention to a neuropathic pain-inducing rat and observing that the threshold for pain is significantly increased compared to the vehicle group. was confirmed (see Test Example 4).

In addition, when the compound of the present invention is administered at a high dose, it is observed that the analgesic effect is maintained until 3 hours after administration, thereby alleviating chronic mechanical allodynia caused by the compound of the present invention pressing on the cauda equina. It was confirmed that there was an effect (see Test Example 4).

In another embodiment of the present invention, an experiment was performed to observe through RT-PCR whether the production of inflammatory mediators was inhibited using rats administered with celecoxib, an anti-inflammatory agent, or a compound according to the present invention, and pro-inflammatory cytokines. As a result of performing an experiment to observe whether the generation of Cain and PGE ₂ is inhibited through ELISA, TNF-α, IL-1β, IL-6, and iNOS The expression of mRNA was significantly reduced, and PGE ₂ production in cauda equina was significantly reduced, so that the compound of the present invention had a preventive or alleviating effect on inflammation caused by pressing cauda equina. It was confirmed that there is (see Test Example 5).

The above results demonstrate the utility of the compounds of the present invention for alleviating chronic mechanical allodynia or inflammation, which is a symptom of spinal cord injury or spinal stenosis.

The pharmaceutical composition according to the present invention is 3-(4-chlorophenyl)-4-(4-aminosulfonyl-phenyl)-1-methyl- 1H -pyrrole-2,5-dione or a pharmaceutically acceptable It includes a salt as an active ingredient, and may also include a pharmaceutically acceptable carrier.

In the preparation of the medicament, the content of the compound of the present invention or a pharmaceutically acceptable salt thereof varies depending on the form of the medicament, but is preferably in a concentration of 0.01 to 100% by weight. The pharmaceutically acceptable carrier is commonly used in formulation, and includes, but is not limited to, saline, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, and the like. It does not, and may further include other conventional additives such as antioxidants and buffers, if necessary. In addition, diluents, dispersants, surfactants, binders, lubricants, etc. may be additionally added to form an injectable formulation such as an aqueous solution, suspension, emulsion, or the like, pill, capsule, granule, or tablet. Regarding suitable pharmaceutically acceptable carriers and formulations, formulations can be preferably made according to each component using the method disclosed in Remington's literature. The pharmaceutical composition of the present invention is not particularly limited in the formulation, but may be formulated as an injection, an inhalant, an external preparation for skin, or an oral intake.

The pharmaceutical composition of the present invention may be administered orally or parenterally (eg, intravenously, subcutaneously, dermally, nasally, or applied to the respiratory tract) according to a desired method, and the dosage may vary depending on the patient's condition and body weight, disease Although it varies depending on the degree, drug form, administration route, and time of administration, it may be appropriately selected by those skilled in the art.

The composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type, severity, and drug activity of the patient. , sensitivity to drug, administration time, administration route and excretion rate, duration of treatment, factors including concomitant drugs, and other factors well known in the medical field. The composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple. In consideration of all of the above factors, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the composition according to the present invention may vary depending on the age, sex, and weight of the patient, and generally 0.001 to 150 mg, preferably 0.01 to 100 mg per kg of body weight, is administered daily or every other day, or 1 It can be administered in divided doses 1 to 3 times a day. However, since the administration route, the severity of spinal cord injury or spinal canal stenosis, sex, weight, age, etc. may increase or decrease, the dosage is not intended to limit the scope of the present invention in any way.

On the other hand, as another aspect of the present invention, the present invention provides a method for preventing, controlling or treating spinal cord injury or spinal stenosis comprising administering the pharmaceutical composition to an individual.

In the present invention, "subject" means a subject in need of a method for preventing, controlling or treating a disease, and more specifically, a human or non-human primate, mouse, rat, dog, cat , refers to mammals such as horses and cattle.

Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

[Example]

Example 1. Experimental preparation and experimental method

1-1. ^One H NMR Spectra and ¹³ C NMR Spectra

^{The instrument used for 1} H NMR Spectra and ¹³ C NMR Spectra in this experiment is a Bruker Avance DXR 400 (400 MHz) Spectrometer.

In addition, chemical shift (δ) is expressed in ppm using tetramethylsilane (TMS) as an internal reference material, coupling constant (J) is expressed in Hertz (Hz), and signal multiplicity is s (singlet) , d (doublet), t (triplet), q (quartet), br (broad), m (multiplet), dd (doulet of doublet).

*In addition, Low-Resolution and High-Resolution Mass Spectra (FABMS, FAB+ energy; 6kev, Emission Current: 5mA, Acceleration Voltage: 10kV) was performed using a JMS-700 Mass Spectrometer (JEOL, JAPAN).

Meanwhile, for TLC, a glass plate coated with silica gel (E.Merck Kiesegel 60F ₂₅₄ layer Thickness 0.25 mm) was used. (phosphomolybdic acid) (PMA) 5 % ethanol solution, p-anisaldehyde 5 % ethanol solution, or ninhydrin 5 % ethanol solution was used as a coloring agent. .

In addition, the silica gel of flash column chromatography used to separate the organic mixture was Merck Kiesegel 60Art 9385 (230-400 mesh).

Finally, the reagents necessary for the reaction were mainly purchased from companies such as Sigma-Aldroch, TCI, Acros or Fluka, and the solvent required for purification was purified by a known method and used.

1-2. Animals and ethics statement

For the experiment according to the present invention, a total of 167 male Spraque-Dawley rats (250 to 270 g, Samtako, Osan, Korea) were used. Animals were stored in an environment of room temperature (23 ± 1 °C) and humidity (60 ± 10%) under a 12 h light/dark cycle (light on 07:30-19:30 h) with free access to water and food. . Rats were housed one at a time in cages (410 X 282 X 153 mm, clear polycarbonate) lined with aspen shaving bedding, on a commercial diet (5L79, PMI Nutrition International, St Louis, MO), on a commercial standard diet (Lab Diet 5L791 Purina). Mills, Richmond, IN). All animal experiments were performed in accordance with the guidelines of the Animal Protection Committee of Kyung Hee University (Permission No.: KHUASP(SE)-15-006), and the ethical issues of the International Association for Pain Research were complied with.

1-3. Surgery and cauda equina compression

Cauda equina compression was induced based on a previous report [PLoS One (2013) e56580, Ma et al.].

The surgical procedure of the rat is as shown in FIG. 1 . More specifically, the rats were anesthetized by administering chloral hydrate (500 mg/kg) by intraperitoneal injection, the back region was shaved, and the L4-S2 vertebrae were exposed.

Next, the ligamenta flava between L4 and L5 is removed, and a trapezoidal silicone block (1.00 mm length X 1.2 to 1.3 mm width X 1.00 mm height) is inserted into the epidural space to insert the L5 and L6 vertebrae into the epidural space. It was placed on a plate and the dural sac was destroyed.

On the other hand, in the case of the sham control group (sham surgery group), only posterior opening and perforation were performed without inserting a silicone block in the rat.

During the surgical procedure, the body temperature of the rat was maintained at 37 ± 0.5 °C using a heating pad (Biomed S.L., Alicante, Spain). After the above injury, the muscles and skin were occluded and the rats were placed in a temperature and humidity controlled chamber overnight.

The operated rats received subcutaneous supplemental fluid (5 ml, lactated ringer) and antibiotics (gentamicin, 5 mg/kg, intraperitoneal injection) once a day for 5 days. Body weight and food and water leftovers were recorded every morning for all animal models.

1-4. behavior assessment

Locomotor activity was measured using a rotarod system (Rota ROD-R V2.0, B.S. Technolab.Inc).

More specifically, the rats were placed on a rod with increasing speed from 4 rpm to 40 rpm (accelerating to 1 rpm per 5 seconds). The time the rats walked until they fell off the rod was measured three times for each animal. Rats were acclimatized to the rod for 3 minutes at a constant speed of 4 rpm prior to the above measurements. The interval between experiments was set to 20 minutes. For statistical analysis, the average value of three clinical trials was calculated.

Mechanicl allodynia was a probing response to a corrected von Frey filament stimulus, and was evaluated by the paw withdrawal threshold (PWT). Pain behavior tests were performed by experienced investigators who were unaware of the experimental conditions.

1-5. drug injection

Only rats inducing chronic mechanical allodynia (2.5-4.0 g) between 350 and 380 g on the 28th day after compression of Cauda equina were selected as the experimental group. The rats were randomly assigned to each experimental group treated with vehicle, celecoxib, and a compound according to the present invention.

Celecoxib (Sigma, St. Louis, MO) or a compound according to the present invention is dissolved in methyl pyrrolidone: Tween-80: saline (1: 1: 8, 100 μl) and injected intraperitoneally. 2, 5 or 10 mg/kg. The vehicle group was injected with 1-methyl-2-pyrrolidone (1-Methyl-2-pyrrolidone: Tween-80: saline (1: 1: 8)) at an equal dose at the time point.

1-6. tissue preparation

At the peak effect (30 minutes after drug injection), rats were anesthetized by injection of chloral hydrate (500 mg/kg) and perfused with 0.1 M PBS (pH 7.4), followed by 4% paraformaldehyde in PBS. was perfused with a solution containing

To make frozen sections, the segments were embedded in OCT and cut into sections at 10 °C using a cryostat (CM1850; Leica, Wetzlar, Germany).

For molecular level analysis, rats were perfused with 0.1 M PBS, and a 20 mm cauda equina section centered at the injured site was isolated and frozen at -80 °C until use.

1-7. Immunohistochemistry

Frozen sections were immunohistochemically treated with an antibody to ED-1 (CD68, 1:200, Serotec, Raleigh, NC) and an antibody to COX-2 (1:100, Abcam, MA). Fluorescence signals were detected by fluorescence microscopy (BX51, Olympus, Japan), and measurement of signal colocalization was performed using MetaMorph software (Molecular devaices, Sunnyvale, CA).

1-8. Western blot

Total proteins from cauda equina sections containing the compression site were prepared and western blot analysis was performed. The primary antibodies used for western blot were as follows; COX-2 (1:1000, Abcam) and β-tublin (1:3000, Sigma).

Quantification of bands was performed using AlphaImager software (Alpha Innotech Coperation, San Leandro, CA).

1-9. RNA isolation and real-time RT-PCR

of tnf-α, il-1β, il-6, inos and gapdh RT-PCR was performed. Primers of each sequence are shown in Table 1 below (5'->3').

division designation forward reverse One TNF-α 5'-CCC AGA CCC TCA CAC TCA GAT-3' 5'-TTG TCC CTT GAA GAG AAC CTG-3' 2 IL-1β 5'-GCA GCT ACC TAT GTC TTG CCC GTG-3' 5'-GTC GTT GCT TGT CTC TCC TTG TA-3' 3 IL-6 5'-AAG TTT CTC TCC GCA AGA TAC TTC CAG CCA-3' 5'-AGG CAA ATT TCC TGG TTA TAT CCA GTT-3' 4 COX-2 5'-CCA TGT CAA AAC CGT GGT GAA TG-3' 5'-ATG GGA GTT GGG CAG TCA TCA G-3' 5 iNOS 5'-CTC CAT GAC TCT CAG CAC AGA G-3' 5'-GCA CCG AAG ATA TCC TCA TGA T-3' 6 GAPDH 5'-AAC TTT GGC ATT GTG GAA GG-3' 5'-GGA GAC AAC CTG GTC CTC AG-3'

1-10. PGE ₂ level measurement The level _{of PEG 2} in cauda equina fibers was analyzed using a PEG ₂ ELISA kit (Monoclonal, Cayman Chemical Ann Arbor, MI) according to the manufacturer's instructions.

1-11. statistical analysis

Data are presented as mean ± SD or SEM.

For comparison between experimental groups, statistical significance was evaluated using the unpaired student t test. Multiple comparisons between groups were performed by one-way ANOVA.

Some behavioral assessment scores were analyzed by repeated measures ANOVA. In Dunnett's case, comparison was used with Posthoc anlaysis.

Group size was expressed as the number of animals in each group. Statistical significance was accepted as p < 0.05. All statistical analyzes were performed using SPSS 15.0 (SPSS science, Chicago, IL).

Example 2. 3-(4-Chlorophenyl)-4-(4-aminosulfonyl-phenyl)-1-methyl-1 as a compound of the present invention H -Preparation of pyrrole-2,5-dione

2-1. Preparation of 2-(4-(chlorosulfonyl)phenyl)acetic acid

_{ClSO 3} (20 ml) was cooled in an ICE bath. After slowly adding phenylacetic acid (5.00 g, 36.72 mmol) to this, the ice bath was removed, and the temperature of the solution was raised to room temperature and stirred for 12 hours. Upon completion of the reaction, the solution was slowly dropped into ice water to remove _{remaining ClSO 3 H.} This was filtered to obtain 2-(4-(chlorosulfonyl)phenyl)acetic acid as a white solid product (7.84 g, 91 %).

¹ H-NMR (400 MHz, DMSO- d ₆ ) δ: 11.6 (1H, s), 7.56 (2H, d, J = 8.4 Hz), 7.23 (2H, d, J = 8.4 Hz), 3.58 (2H, s).

2-2. Preparation of 2-(4-sulfaammoylphenyl)acetic acid

In an ICE bath, 2-(4-(chlorosulfonyl)phenyl)acetic acid (2.00 g, 8.55 mmol) obtained in Example 2-1 was dissolved in anhydrous MeOH and cooled. NH ₄ OH (25%) was added dropwise thereto, the ice bath was removed, and the temperature of the solution was raised to room temperature and stirred for 12 hours. Upon completion of the reaction, HCl was added to acidify and stirred under reduced pressure for 12 hours. After completion of the reaction, HCl was added to acidify, and the solvent was removed under reduced pressure. After extraction using EtOAc, the organic layer was dried over anhydrous MgSO ₄ , and the solvent was removed under reduced pressure. The reaction mixture obtained from the organic layer was crystallized using ACN and DCN to obtain 2-(4-sulfaammoylphenyl)acetic acid as a white solid product (1.03 g, 56 %).

¹ H-NMR (400 MHz, DMSO- d ₆ ) δ: 7.75 (2H, d, J = 8.4 Hz), 7.44 (2H, d, J = 8.4 Hz), 7.34 (2H, s), 3.68 (2H, s).

2-3. Preparation of ethyl 2- (4-methylphenyl) -2-oxoacetate

Chlorobenzene (2.00 g, 17.77 mmol) was dissolved in anhydrous DCM under anhydrous conditions and cooled to -5 °C using a low temperature reactor. Anhydrous AlCl ₃ (2.1 g, 16.28 mmol) and ethyl chlorooxoacetate (ethyl chlorooxoacetate, 0.6 ml, 5.43 mmol) were slowly added dropwise thereto. After stirring at 0° C. for 4 hours, the reaction was terminated by adding the product to ice. After extraction with DCM, the organic layer was washed with distilled water. Then, it _{was dried over anhydrous MgSO 4} and filtered under reduced pressure. The solvent was again removed under reduced pressure to obtain ethyl 2-(4-methylphenyl)-2-oxoacetate as a pale yellow liquid product (3.01 g, 80 %).

¹ H-NMR (400 MHz, CDCL ₃ - d ) δ: 7.99 (2H, d, J = 8.4 Hz), 7.49 (2H, d, J = 8.4 Hz), 4.45 (2H, q, J = 7.2 Hz) , 1.43 (3H, t, J = 7.2 Hz).

2-4. Preparation of 2-(4-chlorophenyl)-2-oxoacetic acid

Ethyl 2-(4-methylphenyl)-2-oxoacetate (0.26 g, 1.23 mmol) obtained in Example 2-3 was dissolved in DCM and 2N NaOH was added in excess, followed by stirring at room temperature for 3 hours. Upon completion of the reaction, HCl was added to acidify, and the mixture was extracted with DCM. The organic layer was dried over anhydrous MgSO ₄ , and the solvent was removed under reduced pressure. The reaction mixture obtained from the organic layer was crystallized using DCM and hexane to obtain 2-(4-chlorophenyl)-2-oxoacetic acid as a white solid product (0.14 g, 60%).

¹ H-NMR (400 MHz, CDCL ₃ - d ) δ: 8.31 (2H, d, J = 8.8 Hz), 7.53 (2H, d, J = 8.8 Hz)

2-5. 3-(4-Chlorophenyl)-4-(4-aminosulfonyl-phenyl)-1-methyl-1 H -Preparation of pyrrole-2,5-dione

2-(4-sulfaammoylphenyl)acetic acid (0.10 g, 0.46 mmol) obtained in Example 2-2 and 2-(4-chlorophenyl)-2-oxoacetic acid (0.09 g, 0.46 mmol) _{was dissolved in Ac 2} O, refluxed at 85° C., and stirred for 8 hours. Upon completion of the reaction, the solvent was removed under reduced pressure at high temperature. After the reaction mixture was dissolved in EtOH, CH ₃ NH ₂ (33 %) (0.38 ml, 3.74 mmol) was added in excess and stirred for 18 hours. After the reaction was completed, the solvent was removed under reduced pressure and extracted with EtOAc. The organic layer _{was dried over anhydrous MgSO 4} and the solvent was removed under reduced pressure. The product obtained by column chromatography was crystallized using IPE, and from this, 3-(4-chlorophenyl)-4-(4-aminosulfonyl-phenyl)-1-methyl- 1H -pyrrole as a pale yellow solid product was obtained. -2,5-dione was obtained (0.05 g, 28%).

¹ H-NMR (400 MHz, DMSO- d ₆ ) δ: 7.86 (2H, d, J = 8.4 Hz), 7.56 (2H, d, J = 8.4 Hz), 7.53 (2H, d, J = 8.4 Hz) , 7.45 (2H, br), 7.41 (2H, d, J = 8.4 Hz), 3.04 (3H, s).

¹³ C-NMR (100 MHz, Acetone- d ₆ ) δ: 171.35, 171.29, 145.82, 138.13, 137.06, 136.39, 133.39, 132.58, 131.36, 129.64, 128.37, 127.05, 23.35.

Table 2 below shows the final product 3-(4-chlorophenyl)-4-(4-aminosulfonyl-phenyl)-1-methyl- 1H -pyrrole-2,5-dione and Examples for preparing the same ^{The structure and 1} H-NMR result of the product in each step of step 2 are shown.

division designation structure spectroscopy data Example 2-1. 2-(4(chlorosulfonyl)phenyl)acetic acid

¹ H-NMR (400 MHz, DMSO- d ₆ ) δ: 11.6 (1H, s), 7.56 (2H, d, J = 8.4 Hz), 7.23 (2H, d, J = 8.4 Hz), 3.58 (2H, s). Example 2-2. 2-(4-sulfamoylphenyl)acetic acid

¹ H-NMR (400 MHz, DMSO- d ₆ ) δ: 7.75 (2H, d, J = 8.4 Hz), 7.44 (2H, d, J = 8.4 Hz), 7.34 (2H, s), 3.04 (2H, s). Example 2-3. ethyl 2-(4-chlorophenyl)-2-oxoacetate

¹ H-NMR (400 MHz, DMSO- d ₆ ) δ: 7.99 (2H, d, J = 8.4 Hz), 7.49 (2H, d, J = 8.4 Hz), 4.45 (2H, q, J = 7.2 Hz) , 1.43 (3H, t, J = 7.2 Hz). Example 2-4. 2-(4-chlorophenyl)-2-oxoacetic acid

¹ H-NMR (400 MHz, DMSO- d ₆ ) δ: 8.31 (2H, d, J = 8.8 Hz), 7.53 (2H, d, J = 8.8 Hz) Example 2-5. 3-(4-chlorophenyl)-4-(4-aminosulfonyl-phenyl)-1-methyl-1H-pyrrol-2,5-dione

¹ H-NMR (400 MHz, DMSO- d ₆ ) δ: 7.86 (2H, d, J = 8.4 Hz), 7.56 (2H, d, J = 8.4 Hz), 7.53 (2H, d, J = 8.4 Hz) , 7.45 (2H, br), 7.41 (2H, d, J = 8.4 Hz), 3.04 (3H, s).
¹³ C-NMR (100 MHz, Acetone- d ₆ ) δ: 171.35, 171.29, 145.82, 138.13, 137.06, 136.39, 133.39, 132.58, 131.36, 129.64, 128.37, 127.05, 23.35.

Test Example 1: 3- (4-chlorophenyl) -4- (4-aminosulfonyl-phenyl) -1-methyl-1 H -Biological evaluation of pyrrole-2,5-dione Search for bioactivity

The physiologically active effect of 3-(4-chlorophenyl)-4-(4-aminosulfonyl-phenyl)-1-methyl- 1H -pyrrole-2,5-dione prepared according to Example 2 was When the activity was inhibited by 50%, the concentration value was expressed as IC ₅₀ . On the other hand, the cytotoxicity of the compounds of the present invention was measured by using the MTT assay cell viability (viability) value (IC ₅₀ ).

1-1. LPS-induced PGE of macrophage lines ₂ generative suppression search

RAW 264.7 (mouse macrophage cell line) was purchased from the Korea Cell Line Bank (KCLB) and used in DMEM containing 10% FBS (Foetal Bovine Serum), penicillin (100 units/mL), and streptomycin sulfate (100 μg/mL). Badge (Dulbecco's) modified Eagle's medium) in a humid environment at 37 °C and 5% carbon dioxide.

RAW 264.7 in DMEM medium ^{24 wells were seeded with 1 ml each at 5Х10 5} cells/mL using a cell, left overnight, and then the medium was changed, and then the drug was treated at an appropriate concentration. After incubation for 1 hour, LPS was treated at 1 μg/ml and incubated for 24 hours (or an appropriate time). The supernatant was taken and diluted 5-fold. Add 150 μl of assay buffer to NSB (Non Specific Binding) wells, 100 μl of assay buffer _{was placed in zero standard (B 0} ) wells. Remaining wells were loaded with 100 μl of standard sample and 50 μl of PEG ₂ conjugate (excluding NSB). 50 μl of the PGE2 antibody solution was added and shaken for 2 hours. Each well was aspirated and washed 5 times with wash buffer. 200 μl of pNPP (para-Nitrophenyl phosphate) substrate was added to all wells, stored at room temperature (in the bench) for 1 hour, 50 μl of stop solution was added, and absorbance was measured at 405 nm. _{The amount of PEG 2} production was quantified using the measured absorbance value and the standard curve, _{and the concentration (IC 50} ) inhibiting 50% compared to the group treated with LPS alone was obtained. The results are shown in Table 3 below. In addition, as shown in FIG. 2 , it was confirmed that the IC _{50 value for inhibition of PEG 2} production was measured to be 5.95 nM (positive control: NS-398, 3 μM). This is because 3-(4-chlorophenyl)-4-(4-aminosulfonyl-phenyl)-1-methyl- 1H -pyrrole-2,5-dione has the effect of inhibiting _{LPS-induced PEG 2 biosynthesis.} 8.70 nM (IC ₅₀ ).

1-2. Measurement of cytotoxicity against macrophages

RAW 264.7 (mouse macrophage cell line) was cultured in DMEM medium containing 10% FBS, penicillin (100 units/ml), and streptomycin sulfate (100 μg/ml) in a humid environment at 37°C and 5% carbon dioxide. ^{Cells were collected by centrifugation and scraper, and 1Х10 5} /well was placed in a 96-well plate containing 100 μl of RPMI (Roswell Park Memorial Institute) 1640 medium containing 10% FBS. 3 beta, 4 beta-epoxy-8a-isobutyryloxyguaia-1(10),11,(13)-dien-12.6a-olide was dissolved in methylsulfoxide (DMSO) solvent and all The concentration of DMSO in the test did not exceed 0.1%. After overnight, samples and LPS (1 μg/ml) were added and the plates were incubated for 24 hours. cells were washed once Then, 50 μl of FBS-free medium containing 5 mg/ml of MTT [(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] was added, and cultured at 37°C for 4 hours. After that, the medium is removed and the formazan blue formed in the cells is (formazan blue) was dissolved in 100 μl of DMSO, and absorbance was measured at 540 nm to determine the cytotoxic effect as an IC ₅₀ value. IC ₅₀ refers to the concentration at which the number of cells is reduced by 50% compared to when the compound is not treated.

The cytotoxicity of 3-(4-chlorophenyl)-4-(4-aminosulfonyl-phenyl)-1-methyl- 1H -pyrrole-2,5-dione is shown in Table 3 below.

compound Cell viability
(μM) IC ₅₀ (μM) PGE ₂

>10 0.0087 Celecoxib 0.008

*Reference value: Bioorg. Med. Chem. Lett. (2012), Kaur, J. et al.). Test Example 2: Confirmation of induction of chronic mechanical allodynia using a silicone block

First, it was examined whether chronic neuropathic pain occurs after compression of the cauda equina using the rats that have been operated on in Examples 1-3. Motor and sensory tests were performed pre-operatively and at specific time points between 1 and 28 days post-surgery.

As a result, as shown in Fig. 3a, all animal models were able to walk the rotarod for 289±15 seconds before surgery, but on the 1st day after pressing the cauda equina, the waiting time of the rotarod was 48.2±16 seconds. decreased significantly. In addition, due to the progress of natural recovery, the walking efficiency on the 28th day after compression of the cauda equina slightly increased to 75.2±14 seconds.

On the other hand, as shown in FIG. 3b , the mechanical avoidance threshold (PWT) in response to innocuous mechanical stimuli gradually decreased from the 3rd day after cauda equina compression, and significant tactile allodynia occurred on the 28th day. up to (PWT 3.22 ± 0.6).

In addition, the sham control group (sham surgery group) showed no change in the walking time of rotarod and the tactile withdrawal threshold.

Test Example 3: Confirmation of macrophage infiltration due to cauda equina pressure

Infiltration of inflammatory cells induces the production and secretion of various inflammatory mediators, including _{pro-inflammatory cytokines and PGE 2} , as well as activation of resident immune cells in response to damage to the nervous system. The above inflammatory mediators may promote neuroimmune activation and sensitize primary afferent nerve cells to cause pain hypersensitivity.

Therefore, in order to confirm whether the inflammatory mediators are expressed at the site of macrophage infiltration, an experiment was performed to observe the site of macrophage infiltration.

In addition, an experiment for observing the spatial pattern of macrophages infiltrating in areas other than the spinal cord and cauda equina was performed by immunostaining for ED-1 antibody on the 28th day after compression of the cauda equina. carried out.

As a result, as shown in FIG. 3c, macrophage infiltration was confirmed in the spinal cord tissue and nerve fibers of the cauda equina on the 14th day after compression of the cauda equina.

More specifically, ED-1 positive macrophages were identified in the compression site of the cauda equina, and further infiltrated macrophages in the dorsal nerve fiber bundle of the spinal cord 30 mm away from the uncompressed area and the lesion epicenter ( dorsal funiculus).

Test Example 4: Confirmation of alleviation of chronic mechanical allodynia induced by lumbar spinal canal stenosis of the compound according to the present invention

In order to investigate the effect of the compound of the present invention in chronic mechanical allodynia, celecoxib (2, 5, 10 mg/kg, intraperitoneal injection), an anti-inflammatory agent, was administered to chronic mechanical allodynia-induced rats 28 days after injury.

As a result, as shown in FIG. 4a, it was confirmed that the avoidance response threshold (PWT) for mechanical pain was significantly increased compared to the vehicle group when 30 minutes and 60 minutes passed after the injection of celecoxib (10 mg/kg).

In addition, the compound according to the present invention (2, 5, 10 mg/kg, intraperitoneal injection) was administered to rats inducing chronic mechanical allodynia.

As a result, as shown in FIG. 4B , it was confirmed that the avoidance response threshold (PWT) for mechanical pain increased significantly compared to the vehicle group in a dose-dependent manner when 30 minutes had elapsed after injection of the compound according to the present invention.

In addition, when the compound according to the present invention was administered in a high dose, the analgesic effect was maintained for 3 hours after administration. From these results, it was confirmed that the compound according to the present invention can relieve chronic mechanical allodynia that occurs after compression of cauda equina.

Test Example 5: Confirmation of inhibition of expression of inflammatory mediators of the compound according to the present invention

Using celecoxib and a compound administered with a compound according to the present invention, an experiment was performed to determine whether the production of an inflammatory mediator was inhibited through RT-PCR, and _{whether the production of PGE 2} was inhibited was determined through ELISA An experiment to confirm was performed.

As a result, as shown in FIGS. 4c and 4d , TNF-α, IL-1β, IL-6 and iNOS mRNA levels of TNF-α, IL-1β, IL-6 and iNOS mRNA were treated with celecoxib or a compound according to the present invention in rats inducing chronic mechanical allodynia after 30 minutes had elapsed. It was confirmed that the expression was significantly reduced.

In addition, as shown in Fig. 4e, when compared to the sham control group (sham surgery group), the PEG ₂ production level in the cauda equina of chronic mechanical allodynia-induced rats was significantly increased by the cauda equina compression.

On the other hand, when compared to the vehicle group, when celecoxib or a compound according to the present invention was administered to chronic mechanical allodynia-induced rats, _{it was confirmed that PGE 2} production was significantly reduced.

The description of the present invention stated above is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. There will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

Claims (6)

A pharmaceutical composition for inhibiting inflammation of a spinal cord injury or spinal stenosis comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof.
[Formula Ⅰ]

A pharmaceutical composition for inhibiting _{PEG 2} production, comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof.
[Formula Ⅰ]

A compound represented by formula (I) or a pharmaceutically acceptable salt thereof.
[Formula Ⅰ]

A pharmaceutical composition for alleviating chronic mechanical allodynia comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof.
[Formula Ⅰ]

A pharmaceutical composition for alleviating chronic neuropathic pain caused by compression of a cauda equina, comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof.
[Formula Ⅰ]

A pharmaceutical composition for reducing inflammation or pain associated with spinal stenosis or a symptom of spinal stenosis, comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof.
[Formula Ⅰ]

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