KR101082331B1 - Guggulsterone derivatives, a preparation method thereof, and a use thereof - Google Patents

Abstract

The present invention is a gugulsterone derivative having a prophylactic and therapeutic effect of inflammatory bowel disease excellent in vivo by inhibiting the inflammatory response by interfering with the NF-κB signal transmission system, a preparation method thereof, and a pharmaceutical composition for preventing and treating inflammatory bowel disease It relates to the use as.

Gugulsterone, Inflammatory Bowel Disease

Description

Gugulsterone derivatives, preparation method thereof and use thereof {Guggulsterone derivatives, a preparation method

The present invention relates to a novel gugulsterone derivative, a method for preparing the same, and a use thereof as a pharmaceutical composition for preventing and treating inflammatory bowel disease.

Inflammatory bowel diseases (IBD) are diseases that cause chronic, unexplained inflammation in the intestine and can be classified into ulcerative colitis (UC) and Crohn's disease (CD). In ulcerative colitis, lesions are confined to the large intestine, and bleeding is the main symptom with lesions only in the mucosal layer, whereas Crohn's disease is manifested multiplely in the small intestine and large intestine. , Diarrhea and weight loss are the main symptoms.

Such inflammatory bowel disease is a chronic disease that is not easily treated, and has the property of repeating symptom improvement and recurrence. In addition, there is not yet developed a cure treatment to cure, and the existing treatments also show a number of side effects.

The incidence and prevalence of inflammatory bowel disease, which has been known as a western disease, has recently been rapidly increased in domestic epidemiological studies (Yang SK et al ., Inflamm Bowel Dis 2008; 14: 542-549). Looking at the problems presented in the domestic epidemiologic survey, inflammatory bowel disease in Korea mainly occurs at a young age and lasts for life. In addition, as it increases the probability of developing colorectal cancer along with various complications, it is highly likely that it will be a social problem as well as an increase in medical expenses in the future.

The mechanism of the development of inflammatory bowel disease is not clear, but recent studies have reported the role of inflammatory cytokines such as TNF-α (Pallone F et al ., Aliment Pharmacol Ther 1996; 10 Suppl 2:75). -79; discussion 80; Plevy SE et al ., J Immunol 1997; 159: 6276-628). Meanwhile, nuclear transcription factor-κB (NF-κB), a nuclear transcription factor, plays an important role in the regulation of inflammatory cytokine expression. In other words, NF-κB is an inducible substance that increases in the intestinal inflammatory response and plays an important role in immune and inflammatory responses (Jobin C et al ., Am J Physiol Cell Physiol 2000; 278: C451-462; Neurath MF et al. , Gut 1998; 43: 856-860; Schottelius AJ et al ., Baldwin AS, Int J Colorectal Dis 1999; 14: 18-28). This NF-κB is actually increased in the intestinal laminar propria of Crohn's disease patients (Rogler G et al ., Gastroenterology 1998; 115: 357-369) and is also increased in several animal models. Is observed (Neurath MF et al ., Nat Med 1996; 2: 998-1004). In other words, increased activity of NF-κB increases the expression of genes associated with numerous inflammations, causing inflammation of the intestine. Thus, the association of NF-κB with inflammatory bowel disease suggests that it may be an important target in terms of developing therapeutic agents for inflammatory bowel disease (Jobin C et al ., Inflamm Bowel Dis 2000; 6: 206-213). .

Recently, many plant extracts used in traditional medicine have been able to be synthesized, and it is known that the plant pigment has an anti-inflammatory effect. For example, traditional Indian medicine uses a tree rosin called Commiphora mukul, which has been used for arthritis, obesity, and inflammation. We not only block inflammatory signals by inhibiting the NF-κB signaling pathway in intestinal epithelial cells of the gugulsterone [4,17 (20) -pregnadiene-3,16-dione] in Commiphora mukul extracts. Dextran sulfate sod 윰 (DSS) was found to have an inflammation-reducing effect in the enteritis model of mice, has been registered as Korean Patent Registration No. 10-0825501.

However, in reality, when applying gugulsterone to clinical treatment, not only a large dose is required, but also when administered, it is likely to cause side effects due to absorption in vivo. In order to minimize these side effects, the development of derivatives is required. In other words, if a derivative having higher fat solubility is developed than the existing gugulsterone, it is not absorbed at the time of taking it and can be directly contacted with the inflammatory site on the surface of the intestinal membrane. It can be used effectively. In addition, the synthesis of the derivative has the advantage that can solve the capacity shortage phenomenon due to the extraction of natural substances.

Therefore, the present inventors synthesized a new lipid-soluble gugulsterone derivative, and then confirmed the inhibitory effect in the colonic epithelial cell model and at the same time confirmed the prevention and treatment of enteritis in enteritis animal model by DSS. Gugulsterone derivatives inhibit the inflammatory response by interfering with the NF-κB signal transduction system in vitro , and found to be very effective in preventing and treating inflammatory enteritis in vivo . On the basis of this, the present invention has been completed.

It is an object of the present invention to inhibit the inflammatory response by interfering with the NF-κB signal transduction system, thereby providing a novel gugulsterone derivative having a prophylactic and therapeutic effect in vivo. It is to provide a use as a therapeutic pharmaceutical composition.

In order to achieve the above object, the present invention provides a guggulsterone derivative, a racemate thereof, an optical isomer thereof, and a pharmaceutically acceptable salt thereof represented by Formula 1 below:

[Formula 1]

(In Formula 1, R ₁ is a C3 to C8 linear or branched alkyl group)

In addition,

As shown in Scheme 1 below,

Provided is a method for preparing a gugulsterone derivative of Formula 1 by a Wittig reaction of a compound of Formula 2 with a triphenylphosphonium halide of Formula 3 in the presence of a base and a solvent:

Scheme 1

(In Scheme 1, X is Br, Cl or I, and R ₁ is as described above)

In addition, the present invention is a pharmaceutical composition for preventing and treating inflammatory bowel disease comprising gugugulsterone derivative represented by the formula (1), a racemate thereof, an optical isomer thereof or a pharmaceutically acceptable salt thereof as an active ingredient. To provide.

Gugulsterone derivatives according to the present invention not only inhibit the inflammatory response by interfering with the NF-κB signaling system in vitro , but also exhibit an excellent preventive and therapeutic effect of inflammatory enteritis in vivo .

The present invention is described in more detail below.

The present invention relates to guggulsterone derivatives represented by Formula 1, racemates thereof, optical isomers thereof and pharmaceutically acceptable salts thereof:

[Formula 1]

(In Formula 1, R ₁ is a C3 to C8 linear or branched alkyl group)

Preferably, R ₁ is 1-propyl group, 2-propyl group, 1-butyl group, 2-butyl group, 3-butyl group, 1-pentyl group, 2-pentyl group, 4-pentyl group, 1-hex It is a real group, 2-hexyl group, 5-hexyl group, 1-heptyl group, 2-heptyl group, 4-heptyl group, or 6-heptyl group, and 1-octyl group.

Representative compounds of the invention include compounds as indicated below:

1) (10R, 13S) -17-butylidene-10,13-dimethyl-1,7,8,10,11,12,13,15,16,17-decahydro-2H-cyclopenta [a] Phenanthrene-3 (6H, 9H, 14H) -on

2) (10R, 13S) -17-pentylidene-10,13-dimethyl-1,7,8,10,11,12,13,15,16,17-decahydro-2H-cyclopenta [a] Phenanthrene-3 (6H, 9H, 14H) -on

3) (10R, 13S) -17-hexylidene-10,13-dimethyl-1,7,8,10,11,12,13,15,16,17-decahydro-2H-cyclopenta [a] Phenanthrene-3 (6H, 9H, 14H) -on

4) (10R, 13S) -17-heptylidene-10,13-dimethyl-1,7,8,10,11,12,13,15,16,17-decahydro-2H-cyclopenta [a] Phenanthrene-3 (6H, 9H, 14H) -on

5) (10R, 13S) -17-heptylidene-10,13-dimethyl-1,7,8,10,11,12,13,15,16,17-decahydro-2H-cyclopenta [a ] Phenanthrene-3 (6H, 9H, 14H) -one

More preferably, the gugulsterone derivative of formula 1 according to the present invention is represented by the following formula (10R, 13S) -17-butylidene-10,13-dimethyl-1,7,8,10,11 , 12,13,15, 16,17-decahydro-2H-cyclopenta [a] phenanthrene-3 (6H, 9H, 14H) -one:

[Chemical Formula 5]

The gugulsterone derivative of the present invention represented by Formula 1 may be used in the form of a pharmaceutically acceptable salt, wherein the salt is an acid addition salt formed by a pharmaceutically acceptable free acid. Do.

Organic acids and inorganic acids may be used as the free acid, and hydrochloric acid, bromic acid, sulfuric acid, sulfurous acid, phosphoric acid, etc. may be used as the inorganic acid, and citric acid, acetic acid, maleic acid, fumaric acid, gluconic acid, metalsulfonic acid may be used as the organic acid. , Acetic acid, glycolic acid, succinic acid, tartaric acid, 4-toluenesulfonic acid, galacturonic acid, embonic acid, glutamic acid, citric acid, aspartic acid, and the like can be used.

At this time, the addition salt is a conventional method, that is, after dissolving the compound of Formula 1 in a water miscible organic solvent, such as acetone, methanol, ethanol, or acetonitrile and adding an equivalent or excess of an organic acid or an aqueous acid solution of an inorganic acid It can be prepared by precipitation or crystallization or by evaporation of the solvent or excess acid followed by suction filtration of the dried or precipitated salt.

Since the compound of formula 1 according to the present invention has an asymmetric carbon center in its structure, it may exist as its optical isomer and also as a mixture thereof including racemates. Accordingly, such isomers or mixtures thereof are also within the scope of the present invention. Preferred are racemates in which E -and Z -are mixed.

Gugulsterone derivatives of Formula 1 according to the present invention is prepared by the Wittig reaction of a compound of Formula 2 and a triphenylphosphonium halide of Formula 3 in the presence of a base and a solvent, as shown in Scheme 1 below. do:

Scheme 1

(In Scheme 1, X is Br, Cl or I, and R ₁ is as described above)

Compounds of the formula (II) used as starting materials can be purchased commercially or prepared directly. When the compound of Formula 2 is reacted with a triphenyl phosphonium halide of Formula 3 under a strong base, triphenylphosphine ylide formed as a lysine half of the strong base and the triphenylphosphonium halide is selectively selected from the ketone at the C-17 position. By the Wittig reaction (Guittosterone derivative of Formula 1) is prepared.

The triphenylphosphonium halide of formula (3) is used in 1 to 3 equivalents, preferably 1 to 1.5 equivalents, based on the compound of formula (2).

As the strong base of the reaction, alkali metal hydrides such as lithium hydride, sodium hydride and potassium hydride generally used in the Wittig reaction; organometallic reagents such as n-butyllithium, sec-butyllithium and tert-butyllithium; And alkali metal alcohol base reagents such as sodium methoxide, sodium ethoxide and potassium-t-butoxide. The amount of base used in the reaction is 1 to 1.5 equivalents based on the compound of formula (2).

At this time, the solvent used may be tetrahydrofuran, diethyl ether, hexane, heptane, methanol, ethanol, benzene, toluene, or a mixture thereof.

The reaction is carried out for 30 minutes to 10 hours at -10 ℃ to 120 ℃.

In addition, the present invention provides a pharmaceutical composition for the prevention and treatment of inflammatory bowel disease, which comprises the gugulsterone derivative of Formula 1, its racemate, its optical isomer or its pharmaceutically acceptable salt as an active ingredient.

According to Experimental Example 1 of the present invention, it can be seen that the gugulsterone derivative of Formula 1 significantly decreases IL-8 mRNA expression increased by TNF-α.

According to Experiment 2, the gugulsterone derivative of Formula 1 blocked the phosphorylation of IκBα stimulated by TNF-α to inhibit the expression of inflammatory derivatives by NF-κB.

These results indicate that the gugulsterone derivative of formula 1 according to the present invention inhibits the inflammatory response by interfering with the NF-κB signaling system in vitro .

In addition, according to Experimental Example 3 of the present invention, in the animal model of acute enteritis induced by DSS, the gugulsterone derivative of Formula 1 may be compared with Z-gugulsterone and gugulsterone derivative GG-50B to change the weight and colon of The change in length and disease activity index were all significantly improved, indicating the excellent acute enteritis prevention and treatment effect.

According to Experimental Example 4, the gugulsterone derivative of Formula 1 showed an inflammatory bowel disease treatment effect equivalent to sulfasalazine, prednisolone, which is used as a conventional inflammatory bowel disease treatment.

Through these results, it was confirmed that the gugulsterone derivative of the formula (1) according to the present invention shows an excellent preventive and therapeutic effect of inflammatory bowel disease in vivo ( in vivo ).

The composition of the present invention may further comprise at least one pharmaceutically acceptable carrier in addition to the above-described effective ingredients for administration. Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components, if necessary, as antioxidants, buffers And other conventional additives such as bacteriostatic agents can be added. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to formulate injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Furthermore, it may be preferably formulated according to each disease or component by an appropriate method in the art or using a method disclosed in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA.

The composition according to the present invention can be administered orally or parenterally during clinical administration, and can be used in the form of general pharmaceutical preparations, and when formulated, commonly used fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. Diluents or excipients may be used.

Solid preparations for oral administration may be prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose, lactose or gelatin in the gugulsterone derivative according to the present invention. . In addition to simple excipients, lubricants such as magnesium stearate, talc and the like can also be used. Liquid preparations for oral administration include suspensions, solvents, emulsions or syrups, and various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. can be used in addition to commonly used simple diluents such as water and liquid paraffin. have.

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent or suspension solvent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. may be used, and the bases of the suppositories include Utopepsol, macrogol, Tween 61, Cacao butter, laurin butter, glycerol, gelatin and the like can be used.

The composition of the present invention can be administered parenterally (eg, intravenously, subcutaneously, intraperitoneally or topically) or orally, depending on the desired method, and the dosage is in the range of 0.01 to 1000 mg per kg of body weight. Administration may be divided into several times or several times. In this case, the dosage may be adjusted according to the weight, age, sex, health condition, diet, administration time, administration method, excretion rate and severity of the patient.

Hereinafter, preferred examples and experimental examples are presented to help understand the present invention. However, the following Examples and Experimental Examples are provided only to more easily understand the present invention, and the contents of the present invention are not limited by the Examples.

Example 1 (10R, 13S) -17-butylidene-10,13-dimethyl-1,7,8,10,11,12,13,15,16,17-decahydro-2H-cyclopenta [ a] Preparation of phenanthrene-3 (6H, 9H, 14H) -one

As shown by Scheme 2, the Gugulsterone derivative of Formula 5 (hereinafter, GG-52) was subjected to a Wittig reaction of the compound of Formula 2 and triphenylphosphonium bromide of Formula 4 in the presence of a base and a solvent. Prepared.

After purging nitrogen in the reactor, 500 ml of tetrahydrofuran was added via syringe. Thereafter, 51.8 g (130 mmol) of triphenylphosphonium bromide (Aldrich) of formula 4 was added thereto, and 130 ml (130 mmol) of potassium t-butoxide (Aldrich) was slowly added dropwise thereto, and the reactor temperature was 0. It adjusted to ° C. Stirring at room temperature for 2 hours changed the color of the solution to a deep orange color and stirred for further 2 hours.

Next, a solution of 28.6 g (100 mmol) of the compound of Formula 2 (TCI) in 500 ml of tetrahydrofuran was slowly added dropwise through a syringe, followed by stirring at room temperature for 3 hours. After TLC confirmed that the starting material, the compound of Formula 2, was removed, 32.6 ml of a mixed solution of methanol and water mixed at a volume ratio of 2: 1 was added thereto, stirred for 30 minutes, and then hexane was added thereto. After washing twice with brine, the resulting organic layer was dried over sodium sulfate and filtered to remove the solvent by rotary evaporator. Then purified by silica column with 15% ethyl acetate / 85% hexane developing solution to (10R, 13S) -17-butylidene-10,13-dimethyl-1,7,8 of formula 5 in 50% yield. , 10,11,12,13,15,16,17-decahydro-2H-cyclopenta [a] phenanthrene-3 (6H, 9H, 14H) -one was obtained. As confirmed by HPLC, E- and Z -isomers were mixed at a ratio of 3: 7. The prepared derivatives were stored at -20 ° C, and used in experiments, dissolved in DMSO and diluted with distilled water or PBS. 1 shows the ¹ H NMR spectrum of the obtained GG-52.

Scheme 2

¹ H-NMR (300 MHz, CDCl ₃ ) δ 5.80 (s, 1H), 5.25 (t, J = 7.2 Hz, 1H), 2.55-2.41 (m. 2H), 2.19 -2.01 (m, 6H), 1.92- 1.88 (m. 3H), 1.75-1.62 (m, 4H), 1.54-1.31 (m, 8H), 1.20 (s, 6H), 1.01-0.97 (m. 3H)

Comparative Example 1: (10R, 13S) -10,13-Dimethyl-17-propylidene-1,7,8,10,11,12,13,15,16,17-decahydro-2H-cyclopenta [a Preparation of Phenanthrene-3 (6H, 9H, 14H) -one

Except for using propyltriphenylphosphonium bromide instead of butyltriphenylphosphonium bromide of the general formula (4) as the reactant was carried out in the same manner as in Example 1 (10R, 13S) -10,13-dimethyl-17 -Propylidene-1,7,8,10,11,12,13,15,16,17-decahydro-2H-cyclopenta [a] phenanthrene-3 (6H, 9H, 14H) -one (hereinafter, GG-46) was prepared. 2 shows the ¹ H NMR spectrum of the obtained GG-46.

[Formula 6]

¹ H-NMR (300 MHz, CDCl ₃ ) δ 6.39 (t, J = 7.6 Hz, 1H), 6.17 (s, 1H), 2.45-2.39 (m. 2H), 2.29 -2.19 (m, 2H), 2.02- 1.90 (m. 4H), 1.71-1.52 (m, 6H), 1.20-1.02 (m, 10H), 0.99-0.97 (m. 6H)

Comparative Example 2: (10R, 13S) -17-Butylidene-10,13-dimethyl-1,7,8,10,11,12,13,14,15,17-decahydro-2H-cyclopenta [ a] Preparation of phenanthrene-3,16 (6H, 9H) -dione

2.3 g (2.1 mmol) of selenium dioxide was added dropwise to 100 ml of dichloromethane at 0 ° C, and 15 ml of t-butylperoxide was slowly added dropwise while mixing well, followed by 32.2 g (84 mmol) of the compound of Chemical Formula 5 obtained in Example 1 ) Was dissolved slowly in 85 ml of dichloromethane over 10 minutes, and the temperature was raised to room temperature and stirred for 90 minutes. When the reaction was completed, the resulting organic layer was washed with brine and distilled water, dried over magnesium sulfate and distilled under reduced pressure to obtain a compound of formula 7 in a yield of 93%.

After dissolving 23.9 g (70 mmol) of the compound of Formula 7 in 200 mL of dichloromethane at room temperature, 60 g (700 mmol) of activated manganese dioxide were added at a time and vigorously stirred for 2 hours. Upon completion of the reaction, the reaction mixture was filtered through a short silica gel column to remove manganese dioxide, and the solvent was distilled off under reduced pressure to (10R, 13S) -17-butylidene-10,13-dimethyl-1,7,8 92, yield of 10,11,12,13,14,15,17-decahydro-2H-cyclopenta [a] phenanthrene-3,16 (6H, 9H) -dione (hereafter GG-53) Got it. Figure 3 shows the ¹ H NMR spectrum of the obtained GG-53.

Scheme 3

¹ H-NMR (300 MHz, CDCl ₃ ) δ 6.39 (t, J = 7.8 Hz, 1H), 6.19 (s, 1H), 2.69-2.65 (m. 1H), 2.53 -2.39 (m, 3H), 2.38- 2.19 (m. 3H), 2.11-1.89 (m, 5H), 1.59-1.42 (m, 6H), 1.39-1.30 (m, 2H), 1.12-1.08 (m, 4H), 1.00-0.96 (m. 6H )

Comparative Example 3: (10R, 13S) -10,13-Dimethyl-17-propylidene-1,7,8,10,11,12,13,14,15,17-decahydro-2H-cyclopenta [a ] Phenanthrene-3,16 (6H, 9H) -dione

Except for using propyltriphenylphosphonium bromide instead of butyltriphenylphosphonium bromide of the general formula (4) as the reaction material, was carried out in the same manner as in Comparative Example 2 (10R, 13S) -10,13-dimethyl-17 -Propylidene-1,7,8,10,11,12,13,14,15,17-decahydro-2H-cyclopenta [a] phenanthrene-3,16 (6H, 9H) -dione (hereinafter, GG-50B). 4 shows the ¹ H NMR spectrum of the obtained GG-50B.

[Formula 9]

¹ H-NMR (300 MHz, CDCl ₃ ) δ 6.34 (t, J = 7.8 Hz, 1H), 6.16 (s, 1H), 2.41-2.40 (m. 2H), 2.38 -2.21 (m, 2H), 2.16- 1.82 (m. 6H), 1.61-1.57 (m, 5H), 1.40-1.35 (m, 2H), 1.20-1.06 (m, 8H), 0.99-0.96 (m. 3H)

Comparative Example 4: Z Gugulsterone

Z-Gugulsterone (Calbiochem Inc) (GGS) was used as Comparative Example 4 to compare and evaluate the in vitro and in vivo effects of gugulsterone derivatives on inflammatory bowel disease.

Experimental Example 1: Confirmation of Inhibition of Inflammatory Signal IL-8 mRNA Expression by Gugulsterone Derivatives

The effect of IL-8 mRNA was observed as an inflammatory signal of COGU 205 intestinal epithelial cells prepared above.

1-1. Cell Culture and Treatment

COLO 205, a human colon cell line [KCLB 10222, Korean Cell Line Bank (KCLB), Seoul, Korea], was cultured for 20 to 40 generations and cultured according to the conventional method (Jobin C et al ., J Immunol 1997). 158: 226-234; Sakakima Y et al ., Cancer Gene Ther 2005; 12: 884-889). This cell line was stimulated with TNF-α (10 ng / mL) after 24 hours prior treatment with gugulsterone derivatives.

1-2. RNA Extraction and Real-Time PCR

Cells pretreated with gugulsterone derivatives and stimulated with TNF-α (10 ng / mL) were extracted RNA using Trizol (Invitrogen, Carlsbad, CA, USA). 1 μg of the extracted RNA was reverse transcribed. Later, real-time PCR of human IL-8 was performed using ABI Prism 7000 sequence detection system (Applied Biosystems, Foster City, CA, USA) according to the conventional method (Kim JM et al ., Infect Immun 2007; 75 : 3373-3381). Β-actin was used as a control. All primers were prepared using Primer Express v2.0 by Applied Biosystems. PCR was performed using SYBR Green PCR Master Mix (Applied Biosystems).

Figure 5 compares the degree to which the Gugulsterone derivatives Gugulsterone derivatives GG-46, GG-53, GG-50B and GG-52 according to the present invention inhibits IL-8 mRNA expression induced by TNF-α It is a graph.

Referring to FIG. 5, IL-8 mRNA increased by TNF-α was decreased by pretreatment of the gugulsterone derivatives GG-50B and GG-52. The reduction was proportional to drug dose. On the other hand, when the gugulsterone derivatives GG-46 and GG-53 were treated, the inhibitory effect could not be observed.

Experimental Example 2: Effect of Gugulsterone Derivatives on IκBα Phosphorylation and NF-κB DNA Binding Activity

2-1. Western blot analysis

Western blots were performed to determine whether Gugulsterone derivatives are involved in IκBα phosphorylation associated with NF-κB activation.

Cell lines pretreated with the gugulsterone derivatives in 1-1 were stimulated with TNF-α (10 ng / mL), and then lysed in 1X Laemmli buffer to extract proteins. After SDS-PAGE gel electrophoresis was transferred to nitrocellulose membrane (Hybond ECL, Amersham, Buckinghamshire, England). As the primary antibody, anti-phosphoserine IκBα (Cell Signaling, Beverly, MA, USA), anti-IκBα polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA USA) and anti-β-actin (Santa Cruz Biotechnology) at 1: 1,000, followed by a peroxidase conjugated antibody (peroxidase-conjugated antibody, Biosource) , ECL kit (Amersham, Buckinghamshire, England) after treatment with Kodak X-OMAT film to confirm the expression signal, the results are shown in FIG.

FIG. 6 shows Western blot results performed to compare the effects of gugulsterone derivatives GG-50B and GG-52 on phosphorylation of IκBα stimulated by TNF-α in COLO 205 cell line.

Referring to FIG. 6, the gugulsterone derivative GG-52 according to the present invention reduced the phosphorylation signal of IκBα at both TNF-α stimulation at 10 minutes and 30 minutes, and the gugulsterone derivative GG-50B at 10 minutes of stimulation. Reduced phosphorylation signal.

2-2. Electrophoretic Transfer Shift Black (EMSA)

IκBα phosphorylation in COLO 205 cells means that NF-κB can be translocated into the nucleus. Therefore, in the next experiment, EMSA was performed to evaluate the effect of gugulsterone derivatives on the DNA binding activity of NF-κB in the nucleus.

Cell extracts for the cells of the experiment were made as in the previous method (Jobin C et al ., J Immunol 1997; 158: 226-234.). T4 polynucleotide kinase (5-10 U / μl) in double stranded NF-κB oligonucleotides (5'-AGT TGA GGG GAC TTT CCC AGG C-3 ') (Promega, Madison, Wis., USA) for DNA binding analysis [Γ- ³² P] ATP (3,000 Ci / mmol at 10 mCi / mL) (Amersham) was labeled using an unlabeled oligonucleotide using a G-25 spin column (Boehringer Mannheim Gmbh, Germany). Was removed. 5 μg of the nuclear extract was reacted with 30,000-50,000 CPM of ³² P-labeled DNA for 20 minutes at room temperature, followed by electrophoresis by loading onto 4% unmodified polyacrylamide gel. Thereafter, the gel was dried and subjected to autoradiography, and then the film was developed.

Figure 7 shows the EMSA results carried out to evaluate the effect of gugulsterone derivatives GG-50B and GG-52 on the DNA binding activity of TNF-α stimulated NF-κB in COLO 205 cells.

Referring to FIG. 7, both gugulsterone derivative GG-52 and gugulsterone derivative GG-50B according to the present invention reduced the DNA binding activity of NF-κB in a dose-dependent manner. These results indicate that gugulsterone derivatives GG-50B and GG-52 block IκBα phosphorylation and inhibit the expression of inflammatory derivatives by NF-κB.

Experimental Example 3: Induction of enteritis in mice and confirmation of the prevention and treatment effect of enteritis by gugulsterone derivatives

To investigate the prophylactic and therapeutic effects of gugulsterone derivatives in vivo , acute induction with Dextran Sulfate Sodium (DSS, MP Biochemicals, CA, USA) An enteritis model was used. In statistical analysis, all values were obtained with mean ± standard deviation. Differences between groups were compared by Student's test or Man Whitney U test, and determined to be statistically significant when P value was less than 0.05.

3-1. Experimental animal

Eight-week-old female specific-pathogen free (SPF) C57BL / 6 mice (20-25 g body weight) were purchased from Orient (Seongnam, Korea), 12 hours light and 12 hours Breeding in the absence of light. Experiments were carried out up to 9 weeks after birth, ie 1 week after adaptation. All animal testing procedures passed the deliberation of the Animal Experiment Ethics Committee of Seoul National University Hospital.

3-2. Observation of preventive and therapeutic effects in acute enteritis Model I

According to previous research methods (Lee JY et al. , Int Immunopharmacol 2007; 7: 241-248), 4% DSS solution was mixed with DSS in drinking water and negatively administered for 5 days to induce colitis. Two days before DSS administration, saline was divided into 5 groups for the positive control group and 5 groups for the drug administration group, GG-50B (50 mg / kg qd), GG-50B (200 mg / kg qd), and GG-52 (50 mg / kg qd). ), GG-52 (200 mg / kg qd) and GGS (200 mg / kg qd) were orally administered. 0.5% methyl cellulose was used as the solvent. Body weights, stiffness of feces, and the presence or absence of bloody stool were measured and confirmed daily in the experimental animals. Based on this, according to the previous method (Cooper HS et al. , Lab Invest 1993; 69: 238-249), the disease activity index was measured and compared between the experimental groups. On day 7 of drug administration, the mice were sacrificed and colons were dissected and scored pathologically according to conventional methods (Dieleman LA et al. , Clin Exp Immunol 1998; 114: 385-391). Scoring was based on the severity of inflammation, extent of injury, crypt damage, determined by a skilled pathologist, and the results are shown in Table 1 below.

division % Change in weight Colon length (cm) Disease activity index Control group 100.8 ± 1.4 10.5 ± 0.6 - DSS 81.6 ± 6.4 8.5 ± 0.6 3.0 ± 0.6 DSS + GG-50B
(50 mg / kg) 90.3 ± 5.7 ^* 8.7 ± 0.8 1.0 ± 0.9 ^* DSS + GG-50B
(200 mg / kg) 85.3 ± 4.5 9.4 ± 0.4 ^* 1.8 ± 1.1 DSS + GG-52
(50 mg / kg) 90.9 ± 2.5 ^* 9.4 ± 0.4 ^* 1.8 ± 0.8 DSS + GG-52
(200 mg / kg) 94.3 ± 1.2 ^* 9.5 ± 0.6 ^* 1.3 ± 0.6 ^* DSS + GGS
(200 mg / kg) 89.2 ± 3.8 ^* 9.1 ± 0.3 ^* 2.1 ± 0.3 ^{* *} p <0.05 is compared with DSS only group.

As shown in Table 1, the gugulsterone derivative GG-50B showed an improvement in body weight change and disease activity index (DAI) when administered orally at 50 mg / kg daily for 7 days. There was no effect. In addition, administration of 200 mg / kg showed only improvement in colon length, but no change in weight and DAI could be observed. On the other hand, the gugulsterone derivative GG-52 according to the present invention improved all the weight change, colon length change and DAI value in the positive control group administered only DSS at the dose dose of 50 mg / kg and 200 mg / kg.

FIG. 8 is a photograph showing the results obtained by staining the colon tissues of mice treated with the control group, DSS, DSS, and gugulsterone derivative GG-52 with hematoxylin-eosin (× 100).

Referring to FIG. 8, the large intestine of mice treated with DSS compared to the control group showed complete destruction including internal wounds, loss of epithelial integrity, submucosal edema, and intense inflammatory cell penetration in all layers. Tissue damage was attenuated by administration of the gugulsterone derivative GG-52.

9 is a graph showing inflammation scores by pathological examination of colon tissues of mice treated with the control group, DSS, DSS and gugulsterone derivative GG-50B, DSS and gugulsterone derivative GG-52.

Referring to Figure 9 (A), even in histology, gugulsterone derivative GG-50B did not observe the effect of improving the inflammation score compared to the positive control group only DSS. As a result, it was concluded that the gugulsterone derivative GG-50B had an anti-inflammatory effect in vitro but lacked as an anti-inflammatory agent in vivo .

Referring to FIG. 9 (B), in the histology, gugulsterone derivative GG-52 markedly reduced the inflammation score compared to the positive control, and this preventive effect was proportional to the dose. These results suggest that the gugulsterone derivative GG-52 among the two gugulsterone derivatives observed in laboratory studies may have an anti-inflammatory effect in vivo.

3-3. Observation of preventive and therapeutic effects in acute enteritis model II

A test for acute enteritis model II was performed on GG-52, which was shown to be more effective in acute enteritis model I. According to the previous method (Nam KS et al. , J Microbiol Biotechnol 2007; 17: 1546-1549), 4% DSS was negatively administered for 5 days to induce colitis, followed by saline, gugulsterone derivative GG for 3 days. -52 25 mg / kg qd, 100 mg / kg qd was administered rectally. As with the acute enteritis prevention effect experiment, the disease activity index was measured, and mice were sacrificed on the third day of drug administration and scored according to pathological findings, and the results are shown in Table 2 below.

division % Change in weight Colon length (cm) Disease activity index Control group 103.6 ± 1.8 10.5 ± 0.7 - DSS 86.4 ± 2.9 8.4 ± 0.5 2.6 ± 0.5 DSS + GG-52
(25 mg / kg) 83.9 ± 3.9 8.1 ± 1.1 ^* 1.7 ± 0.8 DSS + GG-52
(100 mg / kg) 97.3 ± 1.2 ^* 8.9 ± 0.3 ^* 0.8 ± 0.4 ^{* *} p <0.05 is compared with DSS only group.

 As shown in Table 2, the weight loss, colon length reduction, and disease activity index (DAI) caused by DSS were all markedly increased by the administration of the gugulsterone derivative GG-52 compared to GGS, the Z-gugulsterone. It was confirmed that the improvement.

Experimental Example 4: Comparison of the Efficacy of Enteritis Treatment with GG-52 and a Previous Drug in DSS-Induced Mice Enteritis Model

In vivo experiments confirmed that gugulsterone derivative GG-52 can be used as a therapeutic agent for inflammatory bowel disease. However, in order to utilize the gugulsterone derivative GG-52 in actual clinical practice, there is a need for the effect to be equal to or greater than that of conventional therapeutics. To this end, we compared the effects of enteritis treatment with sulfasalazine (200 mg / kg) or prednisolone (1 mg / kg) administration group.

According to previous research methods (Lee JY et al ., Int Immunopharmacol 2007; 7: 241-248), colitis was induced by negative administration of 4% DSS solution for 5 days. In the negative control group, saline was orally administered continuously during the experiment. Two days before DSS administration, saline solution was divided into four groups, and the drug group was divided into four groups, sulfasalazine (Sigma Chemical Co., USA; 200 mg / kg qd), prednisolone (Sigma; 1 mg / kg qd), and GG-52 ( 50 mg / kg qd), GG-52 (200 mg / kg qd) was orally administered. 0.5% methyl cellulose was used as the solvent. Mice were weighed daily and their stool firmness and bloody stool were checked daily. Based on this, enteritis prevention effect was compared between the groups in the same manner as described in the acute enteritis prevention observation, and the results are shown in Table 3 below.

division % Change in weight Colon length (cm) Disease activity index Control group 103.6 ± 1.77 10.5 ± 0.71 - DSS 82.1 ± 2.59 ^* 7.5 ± 0.87 3.0 ± 0.0 ^* Sulfasalin
(200 mg / kg) 90.3 ± 2.99 ^{* #} 8.6 ± 1.5 ^* 1.7 ± 0.9 ^{* #} Prednisolone
(1 mg / kg) 89.6 ± 3.40 ^{* #} 8.7 ± 0.46 ^{* #} 1.8 ± 1.3 ^* DSS + GG-52
(50 mg / kg) 85.4 ± 3.92 ^* 8.3 ± 0.39 ^{* #} 2.2 ± 0.2 ^* DSS + GG-52
(200 mg / kg) 92.8 ± 6.01 ^{* #} 9.5 ± 0.7 ^{* #} 1.58 ± 0.8 ^{# *} p <0.05 is compared to the control group, ^# p <0.05 is compared to the DSS only group.

As shown in Table 3, the weight change, colon length change, and disease activity index (DAI) between the GG-52 (50 mg / kg or 200 mg / kg) group and the sulfasalazine or prednisolone group were measured. In comparison, no statistical significance was observed for either of these indicators.

Figure 10 is a graph showing the inflammation scores by pathological examination of colon tissue of mice treated with sulfasalazine, prednisolone, and gugulsterone derivative GG-52.

Referring to FIG. 10, there was no statistical difference in inflammation scores by pathological examination between sulfasalazine, prednisolone, and gugulsterone derivative GG-52. These results suggest that the gugulsterone derivative GG-52 has the same effect as the existing drugs used in the clinical practice for the treatment of inflammatory bowel disease.

Experimental Example 5: Toxicity Test

5-1. Experiment method

Toxicity test of gugulsterone derivatives according to the present invention was confirmed by requesting the professional test institute (Yongin, Korea).

The single oral dose toxicity test was performed using ICR mice, and 2,000 mg / kg, which was 10 times the 200 mg / kg used as a therapeutic dose in enteritis animal models, was administered to mice. In the repeated oral toxicity test, a dose of a derivative was orally administered once a day for 2 weeks, and the following items were examined.

(1) General symptoms observation [SOP-AT-004]

(2) Body weight measurement [SOP-AT-006]

(3) Calculation of Feed Intake [SOP-AT-008]

(4) Water Intake Measurement [SOP-AT-009]

(5) Urinalysis [SOP-AT-010, SOP-BL-008, 009]: Fresh urine collected for 3 to 4 hours in the last week of administration. Measurement was performed using an automatic urine analyzer (CliniTek 100, Bayer). The relevant items are: glucose, bilirubin, ketone body, specific gravity, occult blood, pH, protein, eurobilinogen and nitrite (nitrite) and leukocyte

(6) blood test

A) Hematological test [SOP-BL-005, 014, 015]: Inhaled anesthesia of surviving animals and dead animals in ether during ethereal anesthesia at the time of necropsy. Was carried out. The collected blood was injected into a CBC bottle containing anticoagulant EDTA-2K, and blood counts were measured using CELL-DYN 3700 (Abbot, USA). The inspection items are as follows.

Item unit Way Ⓐ WBC (White blood cell count) 10 ³ / mm ³ Electric resistance method Ⓑ RBC (Red blood cell count) 10 ⁶ / mm ³ Electric resistance method Ⓒ HGB (Hemoglobin conc.) g / ㎗ Cyanmethemoglobin Method Ⓓ HCT (Hematocrit) % b, e Ⓔ MCV (Mean corpuscular volume) fl Electric resistance method Ⓕ MCH (Mean corpuscular hemoglobin) pg Ⓑⓒ Ⓖ MCHC (Mean corpuscular Hb.conc.) g / ㎗ Ⓒⓓ Ⓗ RDW (Red cell distribution with) % Histogram Ⓘ Platelet 10 ³ / mm ³ Electric resistance method Plate MPV (Mean platelet volume) fl Histogram Ⓚ leukocyte differentiation coefficient % Scattered Light Detection

B) Blood biochemical test [SOP-BL-002, 003]: Inject the collected blood into the Vacutainer tube, allow it to coagulate by standing at room temperature for 15-20 minutes, and then centrifuge at 3000 rpm for 10 minutes to AU400 The biochemical tester (Olympus, Japan) was used and the electrolyte was measured using an electrolyte analyzer (644 Na, K, Cl Analyzer, Ciba-Corning, USA).

Item unit Way AST (Aspartate aminotransferase) IU / L IFCC Act Ⓑ ALT (Alanine aminotransferase) IU / L IFCC Act Ⓒ ALP (Alkaline phosphatase) IU / L P-NPP method Ⓓ BUN (Blood urea nitrogen) mg / ㎗ Urease-UV method Ⓔ CRE (Creatinine) mg / ㎗ Jaffe method Ⓕ GLU (Glucose) mg / ㎗ UV method Ⓖ CHO (Total cholesterol) mg / ㎗ Enzyme method Ⓗ PRO (Total protein) g / ㎗ Biuret method CPK (Creatine phosphokinase) IU / l UV-Rate Method B ALB (Albumin) g / ㎗ BCG Act Ⓚ BIL (Total bilirubin) mg / ㎗ Evelyn-Malloy Act Ⓛ TG (Triglyceride) mg / ㎗ Enzyme method Ⓜ IP (Inorganic phosphorus) mg / ㎗ Enzyme method Ⓝ Ca ⁺⁺ (Calcium ion) mg / ㎗ O-CPC method Ⓞ A / G ratio ratio Output as PRO, ALB Ⓟ Na ⁺ (Sodium ion) mmol / L Electrode method ⁺ K ⁺ (Potassium ion) mmol / L Electrode method Ⓡ Cl ^- (Chloride ion) mmol / L Electrode method Ⓐ-ⓞ: measured using a blood biochemical tester (AU400, Olympus, Japan).
Ⓟ-ⓡ: Measured by automated electrolyte analyzer (644 Na, K, Cl Analyzer, Ciba-Corning, USA).

(7) Necropsy [SOP-PA-001]: Animals that have completed blood collection for blood-related examinations were cut by abdominal aorta and posterior vein, and then bleeded to death. It was then recorded on autopsy findings.

(8) Long-term weight measurement [SOP-PA-001]: Heart, lung, thymus, liver, spleen, kidney, adrenal gland, testicle, prostate gland, ovary and uterus at autopsy, and then weighed using electronic balance Both organs were measured.

(9) Long-term fixation [SOP-PA-002]: Organs with abnormal findings in major organs and autopsies were fixed in 10% neutral buffered formalin solution, and histopathological examinations were performed if necessary.

Comparisons between treatment groups in toxicity studies generally used parametric or nonparametric multiple comparison procedures. In other words, in case of parametric multiple comparison, first, one-way batch variance analysis (ANOVA) was used to observe the significance between groups. T-test was performed and Dunnet T-test was performed if homogeneity of variance was not recognized. For general test data observed in urinalysis, non-parametric tests were performed by scale transformation. Kruskal-Wallis'H test using the ranked data was used to confirm the significance between the groups and Mann-Whitney U test. The significance of was tested. All statistical analyzes used SPSS 10.1K, a commercially available statistical package.

5-2. Experiment result

First, a single oral dose toxicity test was performed using ICR mice. To this end, mice were administered 2,000 mg / kg, which is 10 times the 200 mg / kg used as a therapeutic dose in enteritis animal models. As a result, even when the gugulsterone derivative GG-52 (2,000 mg / kg) was administered to the mouse, no animals died. There were no abnormal findings in the general and autopsy findings. Therefore, the minimum lethal dose (MLD) of GG-52 is expected to be over 2,000 mg / kg.

Next, repeated oral administration toxicity test was performed. 1) control group not administered for this purpose; 2) group receiving 222 mg / kg; 3) group administered 666 mg / kg; 4) The group was divided into 2,000 mg / kg and the dose was administered orally once a day for 2 weeks. The test results are as follows.

1) Death Animals: An autopsy of the animals treated with death at 4 days after administration in the 2,000 mg / kg / day female group showed no effect by the test substance.

2) General symptoms: No specific abnormalities were observed.

3) Body weight: Both males and females of the test substance showed a tendency to increase from 1 week of administration. In the male, there was no statistically significant change compared to the excipient control group. In females, the weight gains were significantly increased in the 666 and 2,000 mg / kg / day administration groups compared to the excipient control group (p <0.05, p). <0.01), toxicologically insignificant change.

4) Feed and water intakes: There were no statistically significant changes in feed and water intake groups compared to excipient controls.

5) Urine test: There was a tendency of decreasing pH in the male and female of the test substance GG-52 compared with the excipient control group, but it was not statistically significant. In the 666 and 2,000 mg / kg / day group, males had a significant increase in urinary tract (SG) compared with the excipient control group (p <0.05), but it was not considered a toxicologically harmful change. Glucose decreased significantly in the / kg / day group (p <0.05), but it was considered to be toxicologically insignificant due to a dose-independent change.

6) Hematological test: RDW was significantly decreased in 222 mg / kg / day group compared with excipient control group.

7) Blood biochemical test: A tendency to increase ALP was observed in male and female patients receiving 2,000 mg / kg / day. In the male, there was no statistically significant change compared to the excipient control group, but in the female, the significance was recognized (p <0.05). In addition, total cholesterol (CHO) was significantly increased in the same dose group females compared to the excipient control group (p <0.05). However, they were all considered to be within toxic levels and not toxicologically harmful.

8) Long-term weight: Absolute and relative weight of liver in the male and female treated with 2,000 mg / kg / day showed a significant increase compared to the excipient control (p <0.0.5, p <0.01), 666 and 2,000 mg / kg / day In the female group, the absolute and relative weights of the left and right kidneys increased and tended to increase (p <0.05, p <0.01). However, this was not considered to be a toxicologically harmful change because it was judged that the significant change in the related items was not supported by the kidney and the liver.

9) As a result of visual necropsy findings, there was no change by administration of test substance.

In conclusion, no toxicological changes were observed when GG-52 was administered orally for 2 weeks in ICR male and female mice. Therefore, it was concluded that the no observed adverse effect level (NOAEL) of the test substance GG-52 in the mouse under this test condition was more than 2,000 mg / kg / day in both sexes.

Gugulsterone derivatives according to the present invention can be used as a prophylactic and therapeutic agent for inflammatory bowel disease.

Figure 1 shows the gugulsterone derivative GG-52 ¹ H NMR spectrum.

Figure 2 shows the gugulsterone derivative GG-52 ¹ H NMR spectrum.

Figure 3 shows the gugulsterone derivative GG-52 ¹ H NMR spectrum.

Figure 4 shows the gugulsterone derivative GG-52 ¹ H NMR spectrum.

Figure 5 is a graph showing the degree of inhibition of the expression of gugulsterone derivatives GG-46, GG-53, GG-50B and GG-52 induced by TNF-α.

FIG. 6 shows Western blot results performed to compare the effects of gugulsterone derivatives GG-50B and GG-52 on phosphorylation of IκBα stimulated by TNF-α in COLO 205 cell line.

Figure 7 shows the EMSA results carried out to evaluate the effect of gugulsterone derivatives GG-50B and GG-52 on the DNA binding activity of TNF-α stimulated NF-κB in COLO 205 cells.

Figure 8 is a photograph showing the results observed by staining the colon tissue of the mice treated with the control group, DSS, DSS and gugulsterone derivative GG-52 with hematoxylin-eosin.

9 is a graph showing inflammation scores by pathological examination of colon tissues of mice treated with the control group, DSS, DSS and gugulsterone derivative GG-50B, DSS and gugulsterone derivative GG-52.

Figure 10 is a graph showing the inflammation score by the histopathological examination of the colon tissues of the mice treated with sulfasalazine, gugulsterone derivative of prednisolone Example 1 (GG-52).

Claims (5)

Guggulsterone derivatives represented by Formula 1, racemates thereof, optical isomers thereof and pharmaceutically acceptable salts thereof: [Formula 1]

(In Formula 1, R ₁ is a C3 to C8 linear or branched alkyl group) The method of claim 1, In Formula 1, R ₁ is a gugulsterone derivative that is a 1-propyl group. As shown in Scheme 1 below, A method for preparing a gugulsterone derivative of Formula 1 by performing a Wittig reaction of a compound of Formula 2 with a triphenylphosphonium halide of Formula 3 in the presence of a base and a solvent: Scheme 1

(In Scheme 1, X is Br, Cl or I, and R ₁ is as described above) The method of claim 3, R ₁ in Scheme 1 is a 1-propyl group. A pharmaceutical composition for preventing and treating inflammatory bowel disease, comprising the gugulsterone derivative of claim 1, a racemate thereof, an optical isomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

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