KR20230143963A - Composition for Treating or Preventing Traumatic Brain Injury (TBI) Comprising Verbenone derivatives - Google Patents

Abstract

The present invention relates to a pharmaceutical composition or health functional food for the treatment or prevention of traumatic brain injury comprising a verbenone derivative and a pharmaceutically acceptable salt thereof as an active ingredient. Specifically, the verbenone derivative of the present invention is MMP. Traumatic brain injury can be treated and prevented by alleviating traumatic brain injury through inhibition of -9.

Description

Composition for Treating or Preventing Traumatic Brain Injury (TBI) Comprising Verbenone derivatives}

The present invention relates to a pharmaceutical composition for the treatment or prevention of traumatic brain injury containing a verbenone derivative, and more specifically, to prevent traumatic brain injury by alleviating traumatic brain injury through inhibition of MMP-9 (matrix metalloproteinase-9). It relates to a pharmaceutical composition for the treatment or prevention of traumatic brain injury containing a verbenone derivative capable of treating and preventing traumatic brain injury.

Traumatic brain injury (TBI) refers to damage to brain function due to external force applied to the brain (Control and Prevention, 2015). TBI is a leading cause of death and disability among physical injuries worldwide, injuring 69 million people each year (Rubiano et al., 2015; Dewan et al., 2019). However, despite the urgent need, no treatment has yet been approved by the U.S. Food and Drug Administration (FDA).

Primary damage, which represents mechanical damage to the brain, is followed by secondary damage, which involves complex biochemical events. In secondary damage, excitotoxicity leads to mitochondrial dysfunction and increased oxidative stress, resulting in cell death (Robertson, 2004). Oxidative stress and molecules released from dead cells trigger a neuroinflammatory response, releasing cytokines and recruiting inflammatory cells (Corps et al., 2015). These complex damage mechanisms make therapeutic development difficult and increase the need for multi-targeted treatments (Loane and Faden, 2010; Maas et al., 2010). Among these various injury responses, neuroinflammation plays an important role in chronic neurodegeneration and decreased neurological function after TBI (Kumar and Loane, 2012). A recent study reported an association between long-term cognitive impairment after TBI and chronic neuroinflammation, including chronic microglial activation and chronic elevation of inflammatory cytokines (Simon et al., 2017). Therefore, suppressing neuroinflammation is necessary to prevent chronic neurodegeneration after TBI.

In response to the need for multi-target drugs, verbenone derivatives containing modified styryl moieties with antioxidant, antiexcitotoxic, and antiischemic activities were synthesized (Ju et al., 2013). Among the synthesized berbenone derivatives, SP-8356 (i.e., (1S,5R)-4-((E)-3,4-dihydroxy-5-methoxystryryl)-6,6-dimethylbicylco[3.1.1]hept-3- en-2-one has been shown to have the best antioxidant, anti-excitatory, and anti-ischemic effects, and to date, SP-8356 has been reported to have therapeutic effects in various animal models such as arteriosclerosis, breast cancer, and corneal fibrosis (Pahk et al., 2019; Pahk et al., 2019; Mander et al., 2019; Joung et al., 2020).

Accordingly, the present inventors made efforts to develop a therapeutic agent to suppress the inflammatory response during secondary damage in traumatic brain injury (TBI), and as a result, verbenone derivative compounds inhibited traumatic brain injury through inhibition of MMP-9 (matrix metalloproteinase-9). The present invention was completed after confirming that traumatic brain injury can be treated and prevented by alleviating the damage.

This invention was sponsored by the Ministry of Health and Welfare of the Republic of Korea under the project title “Study on the inhibitory effect and pharmacomechanism of the new concept treatment SP-8356” (KHIDI). ) was conducted with support from the MD-PhD/Medical Scientist Training Program.

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The purpose of the present invention is to regulate inflammatory cytokines and downregulate MMP-9 gene expression and enzyme activity in a controlled cortical impact (CCI) mouse model to reduce TBI-evoked neurobehavioral impairment. The goal is to improve and provide new uses for verbenone derivatives to treat and prevent traumatic brain injury.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the treatment or prevention of traumatic brain injury containing a verbenone derivative represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

In Formula 1,

R ₁ , R ₂ , R ₃ , R ₄ , and R ₅ are each independently a hydrogen atom, a halogen atom, a hydroxy group, a C _1-3 alkyl group, a C _1-3 alkoxy group, an amino group, or a C _1-3 alkylamine. group, C _1-3 alkyldiamine group, C _5-8 aryl group, C _5-8 cyclic group, C _5-8 heteroaryl group, or ego,

X, Y and Z are each independently a carbon atom or an N, O or S atom;

means a double bond or a single bond.

The present invention also provides a health functional food for preventing or improving traumatic brain injury containing the berbenone derivative represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

The compound of formula 1 according to the present invention is a synthetic berbenone derivative and has anti-inflammatory, anti-excitatory toxicity, and antioxidant effects. The compound can improve TBI-induced neurobehavioral disorders through blocking CCI-induced MMP upregulation in a controlled cortical impact (CCI) mouse model. MMP-9 was significantly downregulated by SP-8356 treatment, suggesting that SP-8356 could ameliorate CCI injury through blocking CCI-evoked MMP up-regulation.

Figure 1 Body weight changes after controlled cortical impact (CCI) injury between sham, vehicle, and SP-8356 groups. There was no statistical difference in body weight between the three groups when measured before CCI and 24 hours after CCI injury. Additionally, there was no statistical difference in the degree of weight loss. One-way analysis of variance test was used. Data are expressed as mean ± SD. n = 12 for sham, n = 10 for vehicle and SP-8356 groups.
Figure 2 depicts impaired limb use at baseline and 24 hours after CCI injury among sham, vehicle, and SP-8356 groups. Compared with the vehicle group, impaired limb use in the sham and SP-8356 groups was significantly higher at 24 hours after CCI. Data are presented with box and whisker plots showing all points representing the level (percentage) of impaired limb use for each mouse. n = 12 for sham group; n = 10 for vehicle and SP-8356 groups. Data are expressed as mean ± SD. One-way ANOVA followed by post hoc Tukey test was used. ** p < 0.01 vs. Vehicle. ***p < 0.001 vs. Vehicle.
Figure 3 is a diagram showing quantitative analysis of relative mRNA levels between sham, vehicle, and SP-8356 groups. Increased gene expression of CD11b, TNF-α, TNFR1, TNFR2, IL-6, TGF-β1, and IL-1β following CCI injury is shown. n = 12 for sham group; n = 10 for vehicle and SP-8356 groups. Data are expressed as mean ± SD. One-way ANOVA followed by post hoc Tukey test was used. ** p < 0.05 vs. sham.
Figure 4 is a diagram showing the effect of SP-8356 on the CD147/MMP-9 pathway. MMP-9 was the only gene whose expression level was significantly reduced in the TBI group treated with SP-8356. SP-8356 inhibited MMP-9 activity 24 hours after CCI injury. (A) Quantitative analysis of relative mRNA levels of CD147, MMP-2, and MMP-9 genes. n = 12 for sham group; n = 10 for vehicle and SP-8356 groups. Data are expressed as mean ± SD. One-way ANOVA followed by post hoc Tukey test was used. **p < 0.05 vs. Vehicle. (B) Representative images of MMP-9 gelatin zymography. (C) Quantitative analysis of relative levels of MMP-9 activity in whole tissue lysates. n = 6 for sham, vehicle and SP-8356 groups each. Data are expressed as mean ± SD. One-way ANOVA followed by post hoc Tukey test was used. **p < 0.05 vs. Vehicle.
Figure 5 is a diagram showing the experimental results of a rotarod test of SP-1154, a prodrug of SP-8356. In the mouse CCI injury model, a decrease in fall latency is observed in the rotarod test due to loss of balance immediately after injury, and a gradual increase in exercise time is observed after recovery. At this time, the SP-1154 administration group was observed to maintain a higher overall exercise time for 14 days after CCI injury compared to the control group (vehicle) administration group, but no statistical difference between the two groups was confirmed (A). However, immunochemical staining showed that hyperactivated astrocytes (GFAP) and microglia (Iba-1) in the brain cortex area after CCI injury were significantly decreased in the SP-1154 administration group compared to the control group ( It was confirmed through immunohistochemistry (IHC) (p<0.05; GFAP, p<0.01; Iba-1) (B). The number of animals per group is n=15. The statistical significance of the rotarod test was derived through repeated measures ANOVA, and the drug was administered at 2, 7, 24, and 48 hours after inducing CCI.

Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

Despite the urgent need to develop treatments for traumatic brain injury (TBI), there is still no treatment approved by the U.S. Food and Drug Administration (FDA). In secondary damage that occurs after mechanical damage due to TBI, complex biochemical reactions such as excitotoxicity, oxidative stress, and neuroinflammation occur, and the development of drugs with various mechanisms of action is required to resolve this complex secondary damage. , In TBI, suppressing the inflammatory response during secondary damage is considered an important treatment strategy.

Therefore, we believe that SP-8356 may have therapeutic potential for damage caused by TBI thanks to its inhibitory effects on inflammation, oxidative stress, and excitotoxicity, and that SP-8356 was used in a mouse model of controlled cortical impact (CCI) injury. The effect on TBI-evoked neurobehavioral damage or TBI-evoked neurobehavioral impairment was investigated. In addition, the mechanism of the drug was investigated by examining microglial activation, inflammatory cytokine production, and gene expression related to the CD147/MMP-9 pathway.

The compound of Formula 1 according to the present invention can improve TBI-induced neurobehavioral disorders in a controlled cortical impact (CCI) mouse model through blocking CCI-induced MMP upregulation. MMP-9 was significantly downregulated by SP-8356 treatment, gelatin zymography showed downregulation of MMP-9 activity by SP-8356, and SP-8356 was associated with CCI-induced MMP upregulation ( It was confirmed that CCI damage can be improved by blocking (CCI-evoked MMP up-regulation).

Therefore, the present invention relates to a pharmaceutical composition for the treatment or prevention of traumatic brain injury containing a verbenone derivative represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

In Formula 1, R ₁ , R ₂ , R ₃ , R ₄ , and R ₅ are each independently hydrogen atom, halogen atom, hydroxy group, C _1-3 alkyl group, C _1-3 alkoxy group, amino group, C _{1 -3} alkylamine group, C _1-3 alkyldiamine group, C _5-8 aryl group, C _5-8 cyclic group, C _5-8 heteroaryl group, or ego,

X, Y and Z are each independently a carbon atom or an N, O or S atom;

means a double bond or a single bond.

The present invention evaluated the therapeutic effect of SP-8356 in a mouse model of controlled cortical impact (CCI) injury. In the forepaw use asymmetry test, SP-8356 improved the use ratio of the affected forepaw by about 31.6%, which was reduced due to CCI damage. In quantitative real-time polymerase chain reaction (qRT-PCR), the CCI group had higher levels of MMP-9, transforming growth factor-, and sham molecules than sham molecules. , tumor necrosis factor-α, tumor necrosis factor receptors 1 and 2, interleukin-6, interleukin-1 , gene expression of cluster differentiation 11b (cluster differentiation 11ㅠ, CD11b) was higher. Among these, MMP-9 was the only one whose gene expression was significantly reduced by SP-8356 treatment. In addition, gelatin zymography showed downregulation of MMP-9 activity by SP-8356. In conclusion, SP-8356 alleviated CCI damage by inhibiting the increase in MMP caused by CCI damage, suggesting the potential of SP-8356 as a TBI treatment.

In the present invention, R ₁ , R ₂ , R ₃ , R ₄ , and R ₅ are each independently hydrogen atom, halogen atom, hydroxy group, methyl group, ethyl group, methoxy group, ethoxy group, amino group, C _{5- 6} aryl group, C _5-6 cyclic group, C _5-6 heteroaryl group, or It can be.

In the present invention, R ₁ , R ₂ , R ₃ , R ₄ , and R ₅ are each independently a hydrogen atom, a halogen atom, a hydroxy group, a methyl group, a methoxy group, a phenyl group, a pyrrole group, a pyridine group, or It can be.

In the present invention, the berbenone derivative is

(1 S ,5 R )-4-(4-hydroxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one (3a);

(1 S ,5 R )-4-(4-hydroxy-2-methoxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one (3b);

(1 S ,5 R )-4-(3,4-dihydroxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one (3c);

(1 S ,5 R )-4-(3-bromo-4-hydroxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one (3d);

(1 S ,5 R )-4-(4-hydroxy-2,6-dimethoxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one (3e) ;

(1 S ,5 R )-4-(3,4-dihydroxy-5-methoxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one (3f );

(1 S ,5 R )-4-(3-hydroxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one (3 g);

(1 S ,5 R )-4-(2-hydroxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one (3h);

(1 S ,5 R )-4-(2-hydroxy-4-methoxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one (3i);

(1 S ,5 R )-6,6-dimethyl-4-styrylbicyclo[3.1.1]hept-3-en-2-one (4a);

(1 S ,5 R )-4-(4-fluorostyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one (4b);

(1 S ,5 R )-4-(4-methoxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one (4c);

(1 S ,5 R )-4-(2-(biphenyl-4-yl)vinyl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one (4d);

(1 S ,5 R )-4-(4-(1H-pyrrol-1-yl)styryl)-dimethylbicyclo[3.1.1]hept-3-en-2-one (4e);

(1 S ,5 R )-4-(3,4-dimethoxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one (4f);

(1 S ,5 R )-4-(3,5-dimethoxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one (4 g);

(1 S ,5 R )-4-(2,5-dimethoxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one (4h);

(1 S ,5 R )-4-(5-bromo-2-methoxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one (4i);

(1 S ,5 R )-6,6-dimethyl-4-((E)-2-(pyridin-2-yl)vinyl)bicyclo[3.1.1]hept-3-en-2-one (5a );

(1 S ,5 R )-6,6-dimethyl-4-((E)-2-(pyridin-3-yl)vinyl)bicyclo[3.1.1]hept-3-en-2-one (5b );

(1 S ,5 R )-6,6-dimethyl-4-((E)-2-(pyridin-4-yl)vinyl)bicyclo[3.1.1]hept-3-en-2-one (5c ); and

(2S,2'S)-5-((E)-2-((1R,5S)-6,6-dimethyl-4-oxobicyclo[3.1.1]hept-2-en-2-yl)vinyl) -3-methoxy-1,2-phenylene bis (2-amino-3-methylbutanoate) -dihydrochloride (6), preferably (1 S , 5 R )-4-(3,4-dihydroxy-5-methoxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one (3f) .

[Formula 2]

(Compound 3f)

In the present invention, the composition can improve traumatic brain injury-induced neurobehavioral disorder by regulating inflammatory cytokines and downregulating MMP-9 gene expression and enzyme activity.

In the present invention, SP-8356 does not dissolve well in saline, so it must be used after complete dissolution using an excipient. In addition, the valine ester (SP-1154) of SP-8356, which is a prodrug form of SP-8356, is converted to SP-8356 very quickly and has high water solubility, so it can be administered by dissolving the salt, and has a viscous form. When used by dissolving in hyaluronic acid, it can be applied to corneal damage diseases. Soluble SP-8356 (SP-1154) has similar effects to SP-8356, but is easy to set the dosage and range of administration.

The term "treatment" used in the present invention refers to the improvement or benefit of symptoms of traumatic brain injury by administration of a composition containing the berbenone derivative represented by Formula 1 or a pharmaceutically acceptable salt thereof according to the present invention as an active ingredient. It refers to any action that changes.

The term “prevention” used in the present invention refers to suppressing or delaying the symptoms of traumatic brain injury by administering a composition containing the verbenone derivative represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient according to the present invention. It refers to all actions that are ordered.

As used herein, the term “prodrug” refers to a derivative of a drug molecule that requires conversion to the parent drug through enzymes or hydrolysis to release the active drug within and outside the body. Prodrugs are frequently, but not necessarily, pharmaceutically inactive while the parent drug is being converted.

The compounds of the present invention represented by Formula 1 can be prepared into pharmaceutically acceptable salts and solvates according to methods common in the art. A useful pharmaceutically acceptable salt is an acid addition salt formed by a pharmaceutically acceptable free acid. Acid addition salts are prepared by conventional methods, for example, by dissolving the compound in an excess of aqueous acid and precipitating the salt using a water-miscible organic solvent, such as methanol, ethanol, acetone or acetonitrile. Equimolar amounts of the compound and an acid or alcohol (e.g., glycol monomethyl ether) in water can be heated and the mixture can then be evaporated to dryness, or the precipitated salt can be filtered off with suction.

At this time, organic acids and inorganic acids can be used as free acids. Hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid, etc. can be used as inorganic acids, and methanesulfonic acid, p-toluenesulfonic acid, acetic acid, trifluoroacetic acid, etc. can be used as organic acids. Citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid ( gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, hydroiodic acid, etc. can be used.

Additionally, a pharmaceutically acceptable metal salt can be prepared using a base. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and then evaporating and drying the filtrate. At this time, it is particularly pharmaceutically suitable to produce sodium, potassium or calcium salts as metal salts, and the corresponding silver salts are obtained by reacting an alkali metal or alkaline earth metal salt with an appropriate silver salt (eg, silver nitrate).

Pharmaceutically acceptable salts of the compound having the structure of Formula 1 include salts of acidic or basic groups that may be present in the compound having the structure of Formula 1, unless otherwise indicated. For example, pharmaceutically acceptable salts include the sodium, calcium and potassium salts of hydroxy groups, and other pharmaceutically acceptable salts of amino groups include hydrobromide, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate and dihydrogen. There are phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate (mesylate), and p-toluenesulfonate (tosylate) salts, and methods or manufacturing processes for salts known in the art. It can be manufactured through.

The term “pharmaceutical composition” used in the present invention refers to a product prepared for the purpose of preventing or treating diseases, and can be formulated and used in various forms according to conventional methods. For example, it can be formulated into powders, granules, tablets, capsules, suspensions, emulsions, syrups, etc., and can be formulated and used in the form of external preparations, suppositories, and sterile injection solutions. Specifically, it can be formulated and used in a form suitable for eye administration, for example, eye drops, cream, ointment, gel, or lotion.

The route of administration of the pharmaceutical composition according to the present invention is not limited to these, but is oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, This includes sublingual, instillation, or rectal administration. Oral or parenteral administration is preferred. As used herein, the term “parenteral” includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, instillation, intralesional and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention can also be administered in the form of a suppository for rectal administration. The pharmaceutical composition of the present invention can be administered orally in any orally acceptable dosage form, including, but not limited to, capsules, tablets, and aqueous suspensions and solutions. For oral tablets, commonly used carriers include lactose and corn starch. Lubricants such as magnesium stearate are also typically added. For oral administration in capsule form, useful diluents include lactose and dried cornstarch. When aqueous suspensions are administered orally, the active ingredients are combined with emulsifying and suspending agents. If necessary, sweetening and/or flavoring and/or coloring agents may be added.

The pharmaceutical composition of the present invention varies depending on several factors, including the activity of the specific compound used, age, body weight, general health, gender, diet, time of administration, route of administration, excretion rate, drug formulation, and the severity of the particular disease to be prevented or treated. It can change a lot.

In the present invention, the pharmaceutical composition is formulated or used in combination with one or more drugs selected from the group consisting of calcium channel blockers, antioxidants, glutamate antagonists, anticoagulants, antihypertensive agents, antithrombotic agents, antihistamines, anti-inflammatory analgesics, anticancer agents, and antibiotics. You can use it.

In addition, the present invention relates to a health functional food for preventing or improving traumatic brain injury containing the berbenone derivative represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

The functional food of the present invention can be used in a variety of ways, such as drugs, foods, and beverages for preventing inflammation. The functional food of the present invention includes, for example, various foods, candy, chocolate, beverages, gum, tea, vitamin complexes, health supplements, etc., and can be used in the form of powders, granules, tablets, capsules, or beverages.

The composition containing the compound according to the present invention is formulated in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injection solutions, respectively, according to conventional methods. Carriers, excipients, and diluents that may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, and calcium. Silicates, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate and mineral oil. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain the above compounds with at least one excipient such as cotton, starch, calcium carbonate, or sucrose. Alternatively, it is prepared by mixing lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. . Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurin, glycerogeratin, etc. can be used.

The preferred dosage of the compound of the present invention varies depending on the patient's condition and weight, degree of disease, drug form, administration route and period, but can be appropriately selected by a person skilled in the art. However, for desirable effects, the compound is preferably administered at 0.01 mg/kg to 10 g/kg per day, preferably at 1 mg/kg to 1 g/kg. Administration may be administered once a day, or may be administered in several divided doses. Therefore, the above dosage does not limit the scope of the present invention in any way.

In addition, in another aspect of the present invention, it includes the step of administering to an individual a pharmaceutical composition for the treatment or prevention of traumatic brain injury containing a verbenone derivative represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient. It relates to methods of treating or preventing traumatic brain injury.

In addition, from another aspect of the present invention, it relates to a new use of a pharmaceutical composition for the treatment or prevention of traumatic brain injury containing a verbenone derivative represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[Example]

Preparation Example 1: Preparation of berbenone derivative

Reagent grade (1 S )-(-)-verbenone, 3,4-dihydroxy-5-methoxybenzaldehyde, methylchloromethylether (MOM-Cl), diisopropylethylamine ; DIPEA), potassium hydroxide (KOH), and sodium methoxide (NaOCH3) were purchased commercially, and all reagents and solvents were purchased at high purity and used in additional purification processes except dichlormethane, which was distilled with calcium hydroxide. It was used immediately, and unless otherwise specified, the reaction was performed under a dry nitrogen atmosphere in vacuum-flame dried glassware. Thin-layer chromatography (TLC) uses silica gel (Merck silica gel 60 F ₂₅₄ ) visualized by UV, and column chromatography uses silica gel (E.Merck silica gel, 70-230, 230-400mesh). used.

¹ H-NMR and ¹³ C-NMR spectra were measured at 500 MHz with an instrument (Varian), and chemical shifts were reported in ppm from (TMS) as an internal standard (CDCl ₃ :d7). The migration from TMS (tetramethysilane) used as .26ppm was recorded in ppm and the coupling constant was recorded in hertz. Multiplicity was abbreviated as follows: singlet(s), doublet(d), doublet of doublet(dd), doublet of doublet of doublet(ddd), triplet(t), triplet of doublet(td), doublet of triplet(dt), quartet(q), multiplet(m), and Fetal bovine serum (FBS) was purchased from the company (Hyclone, Logan, UT), and the culture medium (neurobasal medium, NBM) and supplement (B27 supplement) were purchased from the company (Invitrogen, Carlsbad, CA). ) All chemicals and reagents were purchased and used from the company (SigmaAldrich, St. Louis, MO).

The compound of Formula 3f was prepared according to Scheme 1 below.

[Scheme 1]

(One S ,5 R )-4-(3-methoxy-4,5-bis(methoxymethoxy)styryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one (2f) compound manufacture of

400 mg of 3,4-dihydroxy-5-methoxybenzaldehyde was added to 4 mL of dichloromethane, and then 920 mg of diisopropylethylamine was added and cooled. 570 mg of methylchloromethyl ether was added and stirred at room temperature. After adding a small amount of water and separating the layers, the organic layer was dehydrated and concentrated under reduced pressure.

To obtain diene from (1S)-(-)-verbenone by aldol condensation, (1S)-(-)-verbenone 1, 380 mg and 3-meth 620 mg of toxy-4,5-bis(methoxymethoxy)benzaldehyde was added to 6.2 mL of MeOH, 270 mg of KOH was added, stirred at 60°C, and cooled to room temperature. A small amount of water was added, and the organic layer was separated. After dehydration and concentration under reduced pressure, a yellow product was obtained and purified by silica gel column chromatography to obtain (1S,5R)-4-(3-methoxy-4,5-bis(methoxymethoxy)styryl)-6,6. -dimethylbicyclo[3.1.1]hept-3-en-2-one (2f)[(1S,5R)-4-(3-methoxy-4,5-bis(methoxymethoxy)styryl)-6,6- dimethylbicyclo[3.1.1]hept-3-en-2-one(2f)] was obtained (yield 90%).

¹ H-NMR (CDCl _3,500 MHz);

d 6.94(d, J =1.96Hz,1H),6.84(s,2H),6.78(d, J =1.71Hz,1H),5.92(s,1H),5.22(s,2H),5.15(s, 2H), 3.89(s,3H), 3.60(s,3H), 3.52(s,3H), 3.09(t, J =5.75Hz,1H), 2.90(dt, J =9.48,5.53Hz,1H), 2.72(td, J =5.75,1.47Hz,1H),2.10(d, J =9.29Hz,1H),1.57(s,3H),1.00(s,3H);

¹³ C-NMR (CDCl ₃ ,75 MHz); d 203.92, 164.02, 153.63, 151.19, 136.64, 134.76, 132.10, 126.91, 122.43, 108.91,104.88, 98.37, 95.33, 58.19, 57.11, 56.07 , 52.70, 43.78, 39.93, 26.70, 22.11.

(1S,5R)-4-(3,4-dihydroxy-5-methoxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one (((1S ,5R)-4-(3,4-dihydroxy-5-methoxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one), 3f) Preparation of compound

960 mg of compound 2f was added to 5 mL of MeOH, and 630 mg of concentrated hydrochloric acid was added dropwise and stirred. Saturated NaHCO ₃ was added to neutralize the reaction solution, extracted with ethyl acetate, dehydrated, and concentrated under reduced pressure. The final compound was purified and separated by column chromatography to form (1S,5R)-4-(3,4-dihydroxy-5-methoxystyryl)-6,6-dimethylbicyclo as a yellow solid showing the following physical properties. [3.1.1] Hept-3-en-2-one (3f) was obtained and used as a sample in the following experimental example (yield 92%):

mp 168-170℃

[a] ²⁰ _D -158.8000 °(c1.0,MeOH);

¹ H-NMR (CDCl ₃ ,500MHz) d 6.78-6.82(m,3H),6.64(d, J =1.47Hz,1H),5.82-6.01(m,3H),3.92(s,3H),3.10( t, J =5.62Hz,1H),2.91(dt, J =9.35,5.59Hz,1H),2.74(td, J =5.69,1.59Hz,1H),2.12(d, J =9.29Hz,1H), 2.04(s,2H),1.58(s,3H),1.01(s,3H);

¹³ C-NMR (CDCl ₃ ,75 MHz); d204.92,165.07,147.23,144.24,135.54,134.13,128.07,125.

50,121.62,108.63,58.08,56.25,53.12,43.75,40.18,26.78,22.16;

HRMS calculated C18H20O4(M+H)301.1440, measured 301.1453;

HPLC analysis results: (Method 1) 100% (t _R =3.46 min).

Example 1: Effect of Verbenone Derivatives on Traumatic Brain Injury

animal

A total of 50 adult male C57B6/J mice (12 weeks old) were purchased from Koatech (Pyeongtaek, Korea) and raised under specific pathogen-free conditions. All mice were maintained on a 12-hour light/dark cycle with free access to water and food. All mice were randomly assigned to three groups: 1. sham group (N=18), 2. vehicle group (N=16), 3. SP-8356 group (N=16). Of the mice assigned to each group, six were used for gelatin zymography and the remainder were used for qRT-PCR and behavioral testing. All experimental protocols were approved by the Laboratory Animal Management Committee of Seoul National University College of Medicine (Approval Number: 21-0024-S1A0) and were performed in accordance with the National Research Council's Guide for the Care and Use of Laboratory Animals.

Controlled Cortical Impact (CCI) model

Animals were anesthetized using 4% isoflurane in oxygen and placed in a stereotaxic frame. The scalp was incised to expose bregma and lambda. The midpoint between bregma and lambda was marked on the sagittal suture, and then an imaginary line was drawn perpendicular to the sagittal suture toward the right end of the parietal bone. The length of this imaginary line was measured with a flexible paper ruler, and then a circular craniotomy (diameter, 5 mm) was performed centered on the diameter with a drill. Cortical impact injury is Impact One?? Induction was achieved using a stereotactic impactor device (Leica biosystems, Nussloch, Germany). The end of the impactor (diameter, 3 mm) was placed in the center of the exposed brain surface, impacted at a depth of 1.5 mm, speed of 5.0 m/s, and impact duration of 500 ms, and then the scalp was sutured. To relieve pain, acetaminophen (brand name: Tylenol) was dissolved in sterile distilled water at a concentration of 1.5 mg/ml and allowed to drink freely. The control group underwent sham surgery in which all surgical procedures were performed except the induction of cortical traumatic injury.

SP-8356 administration

SP-8356 powder was dissolved in Dimethyl sulfoxide, Ethanol, and Kolliphor® HS 15 and then diluted in 0.9% saline to prepare an injection solution of the desired concentration. 0.2 ml of the prepared SP-8356 solution was injected intraperitoneally at a concentration of 50 mg/kg twice (2 hours and 7 hours after CCI). The test group consists of the SP-8356 administration group, the sham group, and the vehicle group. In the vehicle group, DMSO, Ethanol, and Kolliphor HS 15 solutions without SP-8356 were administered simultaneously at the same administration route and time as above.

tissue preparation

Twenty-four hours after CCI injury, mice were sacrificed after body weight measurement and behavioral assessment. Mice were perfused with 50 mL of normal saline through the left ventricle prior to brain harvest. Mouse brain A 4-mm-thick coronal section of the right hemisphere containing the lesion damaged by CCI was obtained using a stainless steel matrix. The obtained tissue sections were fixed in 0.4% paraformaldehyde or immediately placed in a cryotube and placed in liquid nitrogen to maintain low temperature.

weight

All animals were weighed before and the day after the experiment to compare whether there was a difference in body weight change between groups.

Limb-use asymmetry (cylinder) test

The cylinder test is a useful tool for assessing sensorimotor deficits according to the CCI model of TBI (Baskin et al., 2003). The test assessed limb use asymmetry during vertical navigation in a cylinder (Schallert et al., 2000). Twenty-four hours after CCI injury, mice were placed in a transparent acrylic cylinder and the number of paw touches on the cylinder wall was assessed. The dimensions of the cylinder used in this protocol are 15.0 cm in height, 9.0 cm in inner diameter, 10.0 cm in outer diameter, and 0.5 cm wall thickness. The mouse was placed in an acrylic cylinder with a diameter of 6 cm and filmed with video equipment for 5 minutes before and after the induction of CCI injury. Wall touches were scored for the left and right feet by an observer using recorded video. The number of impaired forelimb uses was calculated as a percentage of total contacts.

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)

The following genes were selected to evaluate gene expression related to microglial activation, inflammatory cytokine production, and CD147/Matrix metalloproteinases-9 (MMP-9) pathway. 1) Cluster of differentiation 11b genes (CD11b), reflecting microglial activation; 2) inflammatory cytokine genes that upregulate MMP-9 activity, IL-1β, TGF-β1, IL-6, TNF-α and TNF-α receptor 1, which regulates overall TNF-α action in corresponding proportions; 2 (TNFR1 and TNFR2) (Watters and O'Connor, 2011); 3) MMP-2, MMP-9 and CD147 (synthetic derivatives of MMPs) (Kaushik et al., 2015). Total RNA was extracted from the harvested brain tissue using TRIzol (Invitrogen), the concentration of RNA was measured, and cDNA was synthesized using a reverse transcription reaction kit (iScript® cDNA synthesis kit, Bio-Rad; Hercules, CA). The sequences of the primers were designed using GenScript (Piscataway, NJ, USA) and are presented in Table 1. mRNA was detected with the CFX96 Touch Real-Time PCR Detection System (Bio-Rad) and quantified with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Livak and Schmittgen, 2001).

Gelatin zymography

에서 48시간 동안 반응시켜 MMP 활성을 유도하였다. 이후, 콜로이드 블루 염색 키트(Invitrogen)를 사용하여 반응이 완료된 겔을 염색하였다. 염색된 겔의 이미지는 ImageJ 오픈 소스 소프트웨어로 정량화되었다(Schneider et al., 2012).Gelatin zymography was used to evaluate the activity of MMP-9 in brain tissue. Harvested brain tissue was harvested from mice sacrificed in the same manner as previously reported and lysed in radioimmunoprecipitation assay buffer (RIPA buufer) containing protease inhibitors. After quantifying the total protein concentration of the brain tissue lysate using the BCA protein assay, 10 μg of protein was mixed with Laemmli sample buffer and loaded on a 10% gelatin zymogram gel, followed by electrophoresis at 200 V for 60 min. The seafood was separated. After separation, the denatured MMP protein in the gel was renaturated and then incubated with development buffer 37.

MMP activity was induced by reacting for 48 hours. Afterwards, the gel upon which the reaction was completed was stained using a colloidal blue staining kit (Invitrogen). Images of stained gels were quantified with ImageJ open source software (Schneider et al., 2012).

statistical analysis

Shapiro-Wilks test of normality followed by one-way analysis of variance (ANOVA) was performed and post hoc Tukey test was used to compare the three groups for parametric data. For non-parametric data, Kruskal-Wallis test was performed and Dwass-Steel-Critchlow-Fligner pairwise comparison was used. Jamovi software version 1.6.23.0 (available at https://www.jamovi.org/) was used for all statistical analyses. A p-value <0.05 was considered statistically significant.

result

weight

Body weight of the sham, vehicle, and SP-8356 groups was measured before CCI injury and the day after CCI injury. The body weight measured before CCI injury was 24.73±0.93 in the sham group, 24.33±0.90 in the vehicle group, and 24.90±1.26 in the SP-8356 group. There was no statistical difference in the body weights of the three groups. The body weight measured the day after CCI was 24.18±1.24 in the sham group, 23.49±0.98 in the vehicle group, and 23.90±1.31 in the SP-8356 group, with no difference. Figure 1 shows the weight of each group at each assessment point and the pattern of decline after CCI injury.

SP-8356 Improved use of impaired limb after CCI

There was no difference in the level of use of the impaired limb between groups at the baseline assessment performed before the CCI injury. However, at the 24-hour assessment after CCI injury, the rate of limb use impairment in the vehicle group was significantly lower than that in the sham group (p-value <0.001). The use of the impaired limb in the SP-8356 group was significantly higher than the vehicle group without SP-8356 treatment (p-value = 0.005) and there was no difference compared to the sham group (p-value = 0.221) (p-value = 0.221). 0.221). Figure 2).

Comparison of inflammation-related gene expression profiles

CCI injury itself upregulated gene expression of CD11b, TNF-α, TNFR1, TNFR2, IL-6, TGF-β1, IL-1β, and MMP-9. However, gene expression of CD147 and MMP-2 was not upregulated by CCI injury (Figures 3 and 4).

SP-8356 downregulated the gene expression and activity of only MMP-9 among the evaluated genes increased by CCI injury. In particular, in gelatin zymography, the size of the band showing MMP-9 activity in the vehicle group was significantly increased compared to the sham group (P value < 0.001), and the density of the band in the SP-8356 group was significantly lower than that of the vehicle group, showing MMP-9 activity. It was confirmed that the activity of 9 was significantly decreased (p value = 0.011, Figure 4).

The present invention confirmed that SP-8356 can improve TBI-induced neurobehavioral disorders in a mouse model of CCI. Additionally, it was confirmed that SP-8356 can regulate inflammatory cytokines in CCI injury. The fact that SP-8356 can improve neurobehavioral disorders in CCI was confirmed by the result that SP-8356 increased the use of the limb damaged by CCI in the limb use asymmetry test, and the regulation of inflammatory cytokines was confirmed by SP-8356 through the MMP-9 gene. This was confirmed through downregulation of expression and enzyme activity.

In the evaluation of sensorimotor function after CCI injury, it was confirmed that SP-8356 improved impaired sensorimotor function by 31.6% the day after injury. CCI injury causes a significant decrease in sensorimotor function in the vehicle group 24 hours after injury. In previous studies, sensory-motor function after CCI deteriorated the most on the day after injury and then gradually recovered (Fox et al., 1998; Henry et al., 2020). Since initial injury severity after TBI is an important predictor of prognosis, reducing functional impairment assessed early is important to shorten the time to functional recovery (Cowen et al., 1995). Considering the relationship between initial injury degree and recovery time, SP-8356 may shorten the recovery period to normal sensorimotor function after CCI. In addition, in the case of the prodrug (SP-1154) being developed to overcome the poor solubility of SP-8356, a behavioral improvement trend (fall latency) was confirmed in the CCI model, but statistical significance was not confirmed. However, a significant decrease in the expression of glial fibrillary acidic protein (GFAP) expressed in the brain tissue of the model was confirmed in the SP-1154 administration group compared to the control group, and the expression of activated microglia was also significant under the SP-1154 administration conditions. It was confirmed that there was a significant decrease. As a result of brain damage caused by CCI, it can be assumed that the impairment of behavioral responses and changes in the neural environment that occur during the brain damage process can be controlled through the prodrug of SP-8356, and the physical and chemical addition of SP-8356 in the future. This indicates that research and development of further improved materials can continue through research (Figure 5)

The present invention discovered downregulation of MMP-9 gene expression and enzyme activity by SP-8356. MMPs are one of the essential proteins upregulated by inflammatory cytokines after TBI (Abdul-Muneer et al., 2016). Among them, MMP-9 regulates blood brain barrier (BBB) integrity and inflammation through dynamic regulation of the extracellular matrix (Abdul-Muneer et al., 2016; George and Geller, 2018). When the BBB is damaged by increased MMP-9 after TBI, circulating inflammatory cells are attracted to the wound site and amplify the inflammatory response (Sulhan et al., 2020). Additionally, increased MMP-9 was associated with tumor necrosis factor-α (TNF-α) (Gearing et al., 1994), interleukin-1β (IL-1β) (Amantea et al., 2016), and transforming growth factor-β ( TGF-β) (Kobayashi et al., 2014). These activated cytokines secreted by microglia, astrocytes, and neurons upregulate MMP-9, exacerbating neuroinflammation (Acarin et al., 2000; Ralay Ranaivo et al., 2011; Takahashi et al., 2014). Therefore, inhibition of MMP-9 in SP-8356 may have reduced the exacerbation of neuroinflammation by inhibiting BBB breakdown and inflammatory cytokines.

Unlike increased MMP-9 gene expression, MMP-2 gene expression was not increased by CCI injury in our study. Upregulated MMP-9 activity has been consistently reported by qPCR and zymography in CCI injury (Lee et al., 2012; Wang et al., 2000). However, MMP-2 activity levels after TBI have been reported to differ depending on age. For example, MMP-2 is predominantly activated compared to MMP-9 in infants, but shows weaker activity than MMP-9 in adults (Sifringer et al., 2007; Wang et al., 2000). A study comparing MMP activity after CCI injury in adult and aged mice found no difference in MMP-2 activity between the two age groups, although aged mice showed higher BBB permeability and higher MMP-9 activity than adult mice. (Lee et al., 2012). This suggests that the regulatory patterns of MMP-2 and MMP-9 after TBI are age-dependent. There was no increase in MMP-2 gene expression or protein activity after CCI, which may be related to the use of 1-week-old adult mice.

CD147 is a transmembrane glycoprotein that induces MMP synthesis and is expressed on a variety of cells, including endothelial cells, monocytes, and platelets (Zhu et al., 2014). In ischemic stroke, upregulated CD147 induces MMP-9, destroying BBB integrity and activating inflammatory cytokines (Jin et al., 2017). However, the effect of TBI on the expression of CD147 is still unknown. To our knowledge, only a single study using a TBI animal mouse model reported that CD147 expression was detected in microvascular endothelial cells and inflammatory cells (Wei et al., 2014). Surprisingly, we showed that there was no difference between CD147 gene expression in the sham, vehicle and SP-8356 groups. Although CCI injury did not increase CD147 gene expression in the examples of the present invention, it is necessary to reconfirm through immunohistochemistry whether CCI injury increases CD147 expression in vascular endothelial cells or inflammatory cells.

In the present invention, the effects of SP-835 on excitotoxicity and oxidative stress were not evaluated. However, excitotoxicity and oxidative stress have a close causal relationship with neuroinflammation in TBI. For example, axons stretched by TBI cause excessive glutamate release and increase excitotoxicity (Dorsett et al., 2017). Excitotoxicity increases oxidative stress and ultimately causes cell swelling and damage, contributing to neuroinflammation (Giza and Hovda, 2001; Roh and Sohn, 2018). In particular, oxidative stress directly induces MMP activation (Abid et al., 2001; Haorah et al., 2007). The role of excitotoxicity and oxidative stress on neuroinflammation raises the question of how much the anti-excitotoxic and antioxidant effects contributed to the neuroprotective effects of SP-8356. Interestingly, SP-8356 has an anti-excitatory effect by inhibiting NMDA-induced intracellular calcium uptake and an antioxidant effect through free radical scavenging (Ju et al., 2013). Although the present invention did not evaluate the anti-excitatory and antioxidant effects of SP-8356 in the CCI model, SP-8356 may have contributed to the inhibition of MMP-9 gene expression and enzyme activity through its anti-excitatory and antioxidant effects.

There are some limitations in this embodiment. First, to evaluate the effect of SP-8356, the CCI model among various TBI models was applied. Because the stereotaxic device immobilizes the head of the mouse in the CCI model, the spreading effects due to the rapid acceleration of TBI may be limited (Marklund and Hillered, 2011). Nevertheless, the CCI model was applied because the pathophysiological mechanisms reported in CCI injury are similar to inflammatory changes in human brain trauma (Edward Dixon et al., 1991). Another reason for applying the CCI model is that the CCI model can apply location coordinates, intensity, speed, and residence time equally to each individual (Osier and Dixon, 2016). The second limitation is that only a single concentration of 50 mg/kg was used to verify the efficacy of SP-8356. Additional studies are needed to optimize the dose, route of administration, and frequency of drug administration. Additionally, additional efforts are needed to increase the solubility of the drug and find ways to exert the drug effect in a more convenient way for patients than intraperitoneal administration.

In summary, we showed that SP-8356 can improve TBI-induced neurobehavioral disorders in a CCI mouse model through blocking CCI-induced MMP upregulation. Considering the antioxidant and anti-excitatory effects of SP-8356 (Ju et al., 2013; Kim et al., 2021; Mander et al., 2019), in the present invention, the anti-inflammatory effect of SP-8356 is effective in treating TBI. It has the potential to be developed as a multi-target drug that can be used for

As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. will be. Accordingly, the actual scope of the present invention will be defined by the claims and their equivalents.

Claims (11)

Pharmaceutical composition for the treatment or prevention of traumatic brain injury comprising a berbenone derivative represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient:
[Formula 1]

In Formula 1,
R ₁ , R ₂ , R ₃ , R ₄ , and R ₅ are each independently a hydrogen atom, a halogen atom, a hydroxy group, a C _1-3 alkyl group, a C _1-3 alkoxy group, an amino group, or a C _1-3 alkylamine. group, C _1-3 alkyldiamine group, C _5-8 aryl group, C _5-8 cyclic group, C _5-8 heteroaryl group, or ego,
X, Y and Z are each independently a carbon atom or an N, O or S atom;
means a double bond or a single bond.
The method of claim 1, wherein R ₁ , R ₂ , R ₃ , R ₄ , and R ₅ are each independently hydrogen atom, halogen atom, hydroxy group, methyl group, ethyl group, methoxy group, ethoxy group, amino group, C _{5 -6} aryl group, C _5-6 cyclic group, C _5-6 heteroaryl group, or A pharmaceutical composition characterized in that:
The method of claim 2, wherein R ₁ , R ₂ , R ₃ , R ₄ , and R ₅ are each independently a hydrogen atom, a halogen atom, a hydroxy group, a methyl group, a methoxy group, a phenyl group, a pyrrole group, a pyridine group, or A pharmaceutical composition characterized in that:
The method of claim 1, wherein the berbenone derivative is
(1 S ,5 R )-4-(4-hydroxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one (3a);
(1 S ,5 R )-4-(4-hydroxy-2-methoxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one (3b);
(1 S ,5 R )-4-(3,4-dihydroxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one (3c);
(1 S ,5 R )-4-(3-bromo-4-hydroxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one (3d);
(1 S ,5 R )-4-(4-hydroxy-2,6-dimethoxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one (3e) ;
(1 S ,5 R )-4-(3,4-dihydroxy-5-methoxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one (3f );
(1 S ,5 R )-4-(3-hydroxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one (3 g);
(1 S ,5 R )-4-(2-hydroxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one (3h);
(1 S ,5 R )-4-(2-hydroxy-4-methoxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one (3i);
(1 S ,5 R )-6,6-dimethyl-4-styrylbicyclo[3.1.1]hept-3-en-2-one (4a);
(1 S ,5 R )-4-(4-fluorostyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one (4b);
(1 S ,5 R )-4-(4-methoxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one (4c);
(1 S ,5 R )-4-(2-(biphenyl-4-yl)vinyl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one (4d);
(1 S ,5 R )-4-(4-(1H-pyrrol-1-yl)styryl)-dimethylbicyclo[3.1.1]hept-3-en-2-one (4e);
(1 S ,5 R )-4-(3,4-dimethoxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one (4f);
(1 S ,5 R )-4-(3,5-dimethoxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one (4 g);
(1 S ,5 R )-4-(2,5-dimethoxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one (4h);
(1 S ,5 R )-4-(5-bromo-2-methoxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one (4i);
(1 S ,5 R )-6,6-dimethyl-4-((E)-2-(pyridin-2-yl)vinyl)bicyclo[3.1.1]hept-3-en-2-one (5a );
(1 S ,5 R )-6,6-dimethyl-4-((E)-2-(pyridin-3-yl)vinyl)bicyclo[3.1.1]hept-3-en-2-one (5b );
(1 S ,5 R )-6,6-dimethyl-4-((E)-2-(pyridin-4-yl)vinyl)bicyclo[3.1.1]hept-3-en-2-one (5c ); and
(2S,2'S)-5-((E)-2-((1R,5S)-6,6-dimethyl-4-oxobicyclo[3.1.1]hept-2-en-2-yl)vinyl) A pharmaceutical composition characterized by at least one selected from the group consisting of -3-methoxy-1,2-phenylene bis(2-amino-3-methylbutanoate)-2-hydrochloride (6).
The method of claim 1, wherein the berbenone derivative is
(1 S ,5 R )-4-(3,4-dihydroxy-5-methoxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one (3f ) A pharmaceutical composition characterized in that.
The pharmaceutical composition according to claim 1, which improves neurobehavioral disorders induced by traumatic brain injury by regulating inflammatory cytokines and downregulating MMP-9 gene expression and enzyme activity.
Health functional food for preventing or improving traumatic brain injury containing a berbenone derivative represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient:
[Formula 1]

In Formula 1,
R ₁ , R ₂ , R ₃ , R ₄ , and R ₅ are each independently a hydrogen atom, a halogen atom, a hydroxy group, a C _1-3 alkyl group, a C _1-3 alkoxy group, an amino group, or a C _1-3 alkylamine. group, C _1-3 alkyldiamine group, C _5-8 aryl group, C _5-8 cyclic group, C _5-8 heteroaryl group, or ego,
X, Y and Z are each independently a carbon atom or an N, O or S atom;
means a double bond or a single bond.
The method of claim 7, wherein R ₁ , R ₂ , R ₃ , R ₄ , and R ₅ are each independently hydrogen atom, halogen atom, hydroxy group, methyl group, ethyl group, methoxy group, ethoxy group, amino group, C _{5 -6} aryl group, C _5-6 cyclic group, C _5-6 heteroaryl group, or A health functional food characterized by:
The method of claim 7, wherein R ₁ , R ₂ , R ₃ , R ₄ , and R ₅ are each independently a hydrogen atom, a halogen atom, a hydroxy group, a methyl group, a methoxy group, a phenyl group, a pyrrole group, a pyridine group, or A health functional food characterized by:
The method of claim 7, wherein the berbenone derivative is
(1 S ,5 R )-4-(4-hydroxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one (3a);
(1 S ,5 R )-4-(4-hydroxy-2-methoxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one (3b);
(1 S ,5 R )-4-(3,4-dihydroxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one (3c);
(1 S ,5 R )-4-(3-bromo-4-hydroxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one (3d);
(1 S ,5 R )-4-(4-hydroxy-2,6-dimethoxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one (3e) ;
(1 S ,5 R )-4-(3,4-dihydroxy-5-methoxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one (3f );
(1 S ,5 R )-4-(3-hydroxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one (3 g);
(1 S ,5 R )-4-(2-hydroxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one (3h);
(1 S ,5 R )-4-(2-hydroxy-4-methoxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one (3i);
(1 S ,5 R )-6,6-dimethyl-4-styrylbicyclo[3.1.1]hept-3-en-2-one (4a);
(1 S ,5 R )-4-(4-fluorostyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one (4b);
(1 S ,5 R )-4-(4-methoxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one (4c);
(1 S ,5 R )-4-(2-(biphenyl-4-yl)vinyl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one (4d);
(1 S ,5 R )-4-(4-(1H-pyrrol-1-yl)styryl)-dimethylbicyclo[3.1.1]hept-3-en-2-one (4e);
(1 S ,5 R )-4-(3,4-dimethoxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one (4f);
(1 S ,5 R )-4-(3,5-dimethoxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one (4 g);
(1 S ,5 R )-4-(2,5-dimethoxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one (4h);
(1 S ,5 R )-4-(5-bromo-2-methoxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one (4i);
(1 S ,5 R )-6,6-dimethyl-4-((E)-2-(pyridin-2-yl)vinyl)bicyclo[3.1.1]hept-3-en-2-one (5a );
(1 S ,5 R )-6,6-dimethyl-4-((E)-2-(pyridin-3-yl)vinyl)bicyclo[3.1.1]hept-3-en-2-one (5b );
(1 S ,5 R )-6,6-dimethyl-4-((E)-2-(pyridin-4-yl)vinyl)bicyclo[3.1.1]hept-3-en-2-one (5c ); and
(2S,2'S)-5-((E)-2-((1R,5S)-6,6-dimethyl-4-oxobicyclo[3.1.1]hept-2-en-2-yl)vinyl) -3-Methoxy-1,2-phenylene bis(2-amino-3-methylbutanoate) -2- dihydrochloride (6) A health functional food characterized by at least one selected from the group consisting of.
The method of claim 7, wherein the berbenone derivative is
(1 S ,5 R )-4-(3,4-dihydroxy-5-methoxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one (3f ) A health functional food characterized by: