WO2024063529A1 - Novel compound, and mri contrast medium to be used in diagnosis and treatment of inflammatory diseases and cancer, containing same - Google Patents

Abstract

The present invention relates to a novel compound and an MRI contrast medium comprising same, the compound having, as a functional group, flavonoids, which are one of natural products having anti-inflammatory, antioxidant and anti-cancer effects. The novel compound of the present invention can be intravenously administered on the basis of having a superior solubility, which differs from that of a conventional insoluble flavonoid, can be expected to exhibit antioxidant, anti-inflammatory and anti-cancer effects even in a small amount and can be used as an T ₁ MRI contrast medium, which targets inflammatory diseases and cancer and enables simultaneous diagnosis and treatment thereof.

Description

New compounds and MRI contrast agents containing them used for diagnosis and treatment of inflammatory diseases and cancer

The present invention relates to a new compound and a gadolinium-based MRI contrast agent. More specifically, it relates to a novel compound that targets inflammatory diseases and cancer and allows simultaneous diagnosis and treatment, and to an MRI contrast agent containing the same.

Flavonoids are bioactive substances found in fruits, vegetables, tea, etc., and have antioxidant and anti-inflammatory effects. Inflammation causes cell damage and promotes the production of ROS, and ROS is a reactive oxygen species that causes an inflammatory response. It is important to control inflammation and ROS. Flavonoids have powerful antioxidant efficacy through direct scavenging of ROS, upregulation of ROS scavenging enzymes, and downregulation of free radicals. Recent studies have shown that flavonoids reduce the inflammatory response by inhibiting the activation of NLRP3 and can be effective in preventing and treating various diseases such as coronary artery disease, metabolic syndrome, and rheumatoid arthritis.

However, flavonoids have very low bioavailability and are difficult to absorb and metabolize for this reason, so they must be consumed in very large amounts to maintain sufficient levels in the body.

In addition, flavonoids have very low solubility in water, so they are mostly administered orally, and orally administered drugs are exposed to a wide variety of environments in digestive pathways such as the mouth, esophagus, stomach, and intestines. Looking at the stomach and intestines, pH has the widest range in the body, from 1.5 to 7. Also, no matter where the drug is absorbed from the stomach to the small intestine, it eventually passes through the liver and then moves to other tissues, so there is a problem in that only a very small amount reaches the target area compared to the drug taken for the first time.

Solubility is a very important issue in the characteristics of drugs, and research and development is needed to increase the solubility of flavonoids and maximize the antioxidant, anti-inflammatory, and anticancer effects of flavonoids even with small amounts.

One object of the present invention is to provide a novel compound that has sufficient solubility to allow intravenous administration and is capable of simultaneously diagnosing and treating inflammatory diseases and cancer, and an MRI contrast agent containing the same.

Another object of the present invention is to provide an anti-inflammatory agent for intravenous administration containing the novel compound.

Another object of the present invention is to provide an antioxidant for intravenous administration containing the novel compound.

The present invention provides a gadolinium complex having galangin, one of the flavonoids with anti-inflammatory, antioxidant, and anti-cancer effects, as a functional group.

A new compound for one purpose of the present invention may be represented by the following formula (1).

(1)

(One)

here,

L is *-(CH ₂ ) _x -A ¹ -(CH ₂ ) _y -A ² -(CH ₂ ) _z -*,

x, y and z are each independently selected as random integers from 0 to 5,

A ¹ and A ² are single bonds, in the group containing *-COO-*, *-CO-*, *-NH-*, *-CH ₂ -*, *-CONH-* and *-O-* Each is one or more independently selected structures,

X is a structure having the following formula (2):

(2)

(2)

Here, R ₁ and R ₂ represent a hydroxy group, and * is a binding site.

In one embodiment, A ¹ may be *-COO-*, and A ² may be *-O-*.

In one embodiment, x may be 1.

In one embodiment, y may be 3.

In one embodiment, z may be 0.

In one example, the compound may be represented by the following formula (3).

(3)

(3)

Here, R ₁ and R ₂ represent a hydroxy group.

In one embodiment, the gadolinium (Gd) may coordinate with one or more water molecules.

In one embodiment, the compound represented by Formula (1) may have a structure in which a flavonoid functional group is bonded to a gadolinium-based compound. Since the compound represented by the formula (1) has sufficient solubility to allow intravenous administration, even in small amounts, the antioxidant, anti-inflammatory, and anticancer effects of the flavonoid itself can be quickly maximized to increase the treatment effect of inflammation and cancer.

In one embodiment, the compounds of the present invention target inflammatory diseases and may have anti-inflammatory activity at the site of inflammation. Due to its high solubility, the compound of the present invention shows a high contrast effect by specifically targeting inflammatory diseases, especially hepatitis, when administered intravenously. Therefore, the compounds of the present invention can be used for diagnosis and treatment of inflammatory diseases. For example, the inflammatory disease may be hepatitis.

In one embodiment, the compounds of the present invention target cancer and may have anticancer activity. Due to its high solubility, the compound of the present invention shows a high contrast effect by specifically targeting serious diseases such as cancer, for example, liver cancer, when administered intravenously. Therefore, the compounds of the present invention can be used for diagnosis and treatment of cancer.

Meanwhile, an MRI contrast agent for other purposes of the present invention may include a compound represented by the above formula (1). The MRI contrast agent of the present invention is T ₁ MRI As a contrast agent, it has excellent contrast around hepatitis, making it easy to target diagnosis and treatment of hepatitis.

In one embodiment, the MRI contrast agent is administered intravenously. Additionally, the MRI contrast agent can be used for targeted diagnosis and treatment of hepatitis.

In one embodiment, the MRI contrast agent has anti-cancer activity and can be used for targeted diagnosis and treatment of cancer. As an example, the compound shows a contrast effect about 1.5 times higher than that of a commercial MRI contrast agent (Gadovist ^® ) for liver cancer cells, HepG2, and can be used for targeted diagnosis and treatment of cancer.

In another embodiment, the present invention can provide an anti-inflammatory agent for intravenous administration, which includes a compound represented by Formula (1), targets an inflammatory site, and has anti-inflammatory activity at the inflammatory site. The anti-inflammatory agent for intravenous administration of the present invention can target the inflammatory site and have therapeutic efficacy. More specifically, it specifically targets hepatitis and exhibits anti-inflammatory activity against hepatitis, so it can have both targeting and therapeutic efficacy against hepatitis.

In another embodiment, the present invention can provide an antioxidant for intravenous administration that includes the compound represented by the formula (1) and has antioxidant efficacy.

The compound having galangin, a flavonoid according to an embodiment of the present invention, as a functional group can be used as an intravenous injection preparation due to its excellent solubility, which is different from existing insoluble flavonoids. Due to this increase in solubility, it is possible to expect increased anti-inflammatory, antioxidant, and anti-cancer efficacy even in small amounts through direct drug delivery, which makes diagnosis and treatment of inflammatory diseases and cancer possible at the same time.

Meanwhile, since the T ₁ MRI contrast agent of the present invention has sufficient solubility to enable intravenous administration, even a small amount can quickly maximize the antioxidant, anti-inflammatory, and anticancer effects of the flavonoid itself, thereby increasing the treatment effect. In particular, for hepatitis, the gadolinium-flavonoid contrast agent of the present invention is a commercial MRI contrast agent (Gadovist) shows higher targeting and contrast effect than that of

When administering flavonoids as therapeutic agents, it is necessary to use micromolar concentrations, because concentrations in the range of 10-100 μM are generally required for antioxidant activity in vivo.

The clinical doses of gadolinium compounds similarly range from 10 to 100 μM, so they can be considered suitable for use with flavonoids. In addition, solubility is a very important issue in drug characteristics, and as a highly water-soluble agent, gadolinium compounds can improve the solubility of flavonoids.

1 is a schematic diagram showing a method for synthesizing a compound according to an embodiment of the present invention.

Figures 2a-2c show the results of HR-FAB-MS analysis of compounds synthesized according to the present invention.

Figures 3a-3c show the results of HPLC analysis of compounds synthesized according to the present invention.

Figure 4 shows the results of confirming the presence of free gadolinium ions in compounds synthesized according to the present invention using an arsenazo III solution.

Figures 5 to 19 are diagrams showing results according to experimental examples of the present invention.

Figure 20 shows the chemical structure of the flavonoid functional group contained in the compound of the present invention.

Figure 21 shows the anti-inflammatory mechanism of Gd-galangin synthesized according to the present invention.

Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings. Since the present invention can be subject to various changes and have various forms, specific embodiments will be illustrated in the drawings and described in detail in the text. However, this is not intended to limit the present invention to a specific disclosed form, and should be understood to include all changes, equivalents, and substitutes included in the spirit and technical scope of the present invention. While describing each drawing, similar reference numerals are used for similar components.

The terms used in this application are only used to describe specific embodiments and are not intended to limit the present invention. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this application, terms such as “comprise” or “have” are intended to designate the presence of features, steps, operations, components, parts, or combinations thereof described in the specification, but are not intended to indicate the presence of one or more other features or steps. , it should be understood that it does not exclude in advance the possibility of the existence or addition of operations, components, parts, or combinations thereof.

Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the technical field to which the present invention pertains. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related technology, and unless explicitly defined in the present application, should not be interpreted in an ideal or excessively formal sense. No.

Compounds according to embodiments of the present invention may have the following formula (1).

(1)

(One)

In the formula (1), the gadolinium ion (Gd ³⁺ ) can form a complex by coordinating with the carboxylate (COO ^- ) group in the formula (1).

In the formula (1), L may be *-(CH ₂ ) _x -A ¹ -(CH ₂ ) _y -A ² -(CH ₂ ) _z -*, and x, y and z are any of 0 to 5. can be each independently selected as an integer, and A ¹ and A ² are a single bond, *-COO-*, *-CO-*, *-NH-*, *-CH ₂ -*, *-CONH-* and *-O-*. * is the binding site.

The L may be a linker connecting the nitrogen and the X in the cyclic structure of the compound. The A ¹ and A ² may determine the functional group determined by the method or the method by which the linker connects the nitrogen and the X in the cyclic structure of the compound. The x, y and z may determine the length of the chain connecting A ¹ and A ² in the linker. In one embodiment, A ¹ may be *-COO-*, and A ² may be *-O-*. In one embodiment, x may be 1. In one embodiment, y may be 3. In one embodiment, z may be 0.

As long as the compound according to the embodiment of the present invention substantially has the structure of Formula (1), bonding or removal of bonding acceptable to those skilled in the art is not excluded from the scope of the present invention. For example, in one embodiment, the gadolinium (Gd) in the compound may coordinate with one or more water molecules.

In the above formula (1), X may have a structure having the following formula (2).

(2)

(2)

Here, R ₁ and R ₂ represent a hydroxy group, and * is a binding site.

In one example, the compound may be represented by the following formula (3).

(3)

(3)

Here, R ₁ and R ₂ represent a hydroxy group.

In one embodiment, because the compound has sufficient solubility to allow intravenous administration, even a small amount can quickly maximize the antioxidant, anti-inflammatory, and anticancer effects of the flavonoid itself, thereby increasing the treatment effect of inflammation or cancer.

In one embodiment, the compound targets inflammatory diseases and may have anti-inflammatory activity at the site of inflammation. Due to its high solubility, the compound of the present invention shows a high contrast effect by specifically targeting inflammatory diseases, especially hepatitis, when administered intravenously. Therefore, the compounds of the present invention can be used for diagnosis or treatment of inflammatory diseases.

In one embodiment, the inflammatory disease may be hepatitis.

In one embodiment, the compound targets cancer and may have anticancer activity. Due to its high solubility, the compound of the present invention shows a high contrast effect by specifically targeting serious diseases such as cancer, for example, liver cancer, when administered intravenously. Therefore, the compound of the present invention can be used for diagnosis or treatment of cancer.

Meanwhile, an MRI contrast agent for other purposes of the present invention may include a compound represented by the above formula (1). The MRI contrast agent of the present invention is T ₁ MRI As a contrast agent, it has excellent contrast around hepatitis, making it easy to diagnose and target diagnosis and treatment of hepatitis.

In one embodiment, the MRI contrast agent is administered intravenously. The MRI contrast agent of the present invention can be particularly used in the diagnosis and treatment of inflammatory diseases or cancer. More specifically, the MRI contrast agent can be used for targeted diagnosis and treatment of inflammatory diseases such as hepatitis.

As another example, the MRI contrast agent has anti-cancer activity and can be used for targeted diagnosis and treatment of cancer. As an example, the compound shows a contrast effect about 1.5 times higher than that of a commercial MRI contrast agent (Gadovist ^® ) for liver cancer cells, HepG2, and can be used for targeted diagnosis and treatment of cancer.

In another embodiment, the present invention can provide an anti-inflammatory agent for intravenous administration, which includes a compound represented by Formula (1), targets an inflammatory site, and has anti-inflammatory activity at the inflammatory site. The anti-inflammatory agent for intravenous administration of the present invention can target the inflammatory site and have therapeutic efficacy. More specifically, it specifically targets hepatitis and exhibits anti-inflammatory activity against hepatitis, so it can have both targeting and therapeutic efficacy against hepatitis.

In another embodiment, the present invention can provide an antioxidant for intravenous administration that includes the compound represented by the formula (1) and has antioxidant efficacy.

Hereinafter, the characteristics of the new compound according to the present invention and the MRI contrast agent containing the same will be described in more detail through specific examples.

Example

Gadolinium complex synthesis

A schematic diagram of the synthesis of a gadolinium complex containing DO3A bound to 7-Hydroxyflavone, chrysin, and galangin is shown in Figure 1. The specific synthesis method is as follows.

Synthesis of compounds 1a-1c

An acetone solution containing flavonoids (1 eq) was added dropwise to an acetone solution of 1,3-dibromopropane (10 eq) and K ₂ CO ₃ (3 eq). Nitrogen the resulting mixture It was stirred at 60°C for 4 hours. After the mixture was cooled to room temperature, it was filtered through a short silica gel column (5% methanol/CH ₂ Cl ₂ , v/v) to remove impurities including inorganic salts. The solvent was removed, and the washing mixture was slowly precipitated at low temperature under CH ₂ Cl ₂ /hexane conditions to separate 1,3-dibromopropane.

Afterwards, it was purified using silica gel column chromatography (0-5% methanol/CH ₂ Cl ₂ , v/v) to obtain 1a-c .

Synthesis of 4-oxo-2-phenyl-4H-chromen-3,5,7-triyl triacetate (2)

Galangin (3,5,7-trihydroxyflavone) (5.4328 g, 20 mmol) was dissolved in THF (200 mL) and triethylamine (34 mL, 240 mmol). Acetic anhydride (24 mL, 240 mmol) was added to the mixture and stirred at room temperature for 24-48 hours. The reaction was monitored by TLC (hexane:ethyl acetate = 1:1) and stirred until the starting material disappeared. The solvent was removed, leaving a crude mixture, which was diluted with ethyl acetate (300 mL) and water (100 mL). The aqueous layer was adjusted to pH 7 with 5% NaHCO ₃ solution, extracted with ethyl acetate, dried over Na ₂ SO ₄ and concentrated. The residue was washed with 10% ethyl acetate/hexane (v/v). Afterwards, product (2) was obtained as a white solid (7.160 g, 90%). mp 146-148℃.

Synthesis of 7-hydroxy-4-oxo-2-phenyl-4H-chromen-3,5-diyl diacetate (3)

Product 2 (7.927 g, 20 mmol) was dissolved in N-methyl-2-pyrrolidone (NMP) (150 mL) under nitrogen and the solution was cooled to 0°C. After adding thiophenol (2.644 g, 24 mmol) while maintaining the temperature, imidazole (0.477 g, 7 mmol) dissolved in NMP was syringed. The mixture was slowly brought to room temperature and reacted for 6-12 hours. The reaction was stirred until TLC (hexane:ethyl acetate=1:1) starting material disappeared. The mixture was then diluted with ethyl acetate and washed with 1M HCl and brine. Next, the organic layer was dried with Na ₂ SO ₄ and concentrated.

The residue was washed with 10% ethyl acetate/hexane and 10% IPA/hexane (v/v). Product (3) was obtained as a white or light yellow solid (13.122 g, 93%). mp 222-224℃.

Synthesis of compounds 6 and 7

5 (1 eq) was dissolved in THF and triethylamine (3 eq) was added. 1a-c (1.2 eq) of THF solution was slowly added and stirred at 40°C for 2 hours. The mixture was then cooled to room temperature and filtered through a short silica gel column (5% methanol/CH ₂ Cl ₂ , v/v) to remove inorganic impurities. Afterwards, 6a-c were obtained by purification using silica gel column chromatography (0-5% methanol/CH ₂ Cl ₂ , v/v).

Next, 6a-c were dissolved in 1,4-dioxane and HCl was added slowly at 0 °C. The reaction was stirred and slowly allowed to reach 40°C. After 12 hours, the solvent was removed, leaving a crude mixture, which was diluted with water and filtered. Then, purification was performed using flash column chromatography on C18 silica (5-95% acetonitrile/water) to obtain 7a-c .

Compound 8 synthesis

7a-c (1 eq) and GdCl ₃ .6H ₂ O (1-1.2 eq) were dissolved in deionized water and the pH of the reaction mixture was adjusted to pH 6 using 1M NaHCO ₃ solution. The mixture was then stirred at room temperature and monitored by TLC and LC-MS. The mixture was centrifuged at 5,000 rpm for 10 minutes, and the supernatant was filtered through a 0.25 μm filter. Next, 8a-c were obtained by purification using flash column chromatography on C18 silica (5-95% acetonitrile/water).

Referring to Figure 1, the hydroxy group at position 7 of the flavonoid ring A is more acidic than the hydroxy groups at other positions, and the highly reactive hydroxy group at position 7 acts as a linker to form 1,3-dibro. 1a-c coupled to mopropane were synthesized. The galangin intermediate protected with an acetyl group (2) reacted with imidazole and thiophenol (PhSH) so that the linker reacted at the same position (3).

Meanwhile, after synthesizing tBu-DO3A-COOH (5), intermediates 6a-6c formed an ester bond with 1a-c under triethylamine and tetrahydrofuran (THF) conditions. Afterwards, tert-butyl and acetyl groups were deprotected with hydrochloric acid (7a-c), and then formed into gadolinium complexes (8a; Gd-flavone, 8b; Gd-chrysin, 8c) in the presence of GdCl ₃ ㆍ6H ₂ O and 1M NaHCO ₃ ; Gd-galangin) was synthesized.

Most reactions provided good yields of over 80%, and it was confirmed that the yield of Gd-galangin could vary (44.41% to 67.95%) depending on reaction time, pH, and temperature.

The synthesized material was confirmed through ¹ H NMR, ¹³ C NMR, HR-FAB-MS, and melting point analysis (Figures 2a-2c). Additionally, the purity of the gadolinium complex (8a-c) was determined based on HPLC analysis (Figures 3a-3c), and the presence of free gadolinium ions was confirmed using arsenazo III solution (Figure 4).

Experimental Example 1: Evaluation of properties of gadolinium-flavonoid complex

Physicochemical properties of synthesized gadolinium complex

The r ₁ and r ₂ relaxivities of Gd-flavone, Gd-chrysin, Gd-galangin and other commercial contrast agents were measured at five concentrations (0.0625, 0.125) in deionized water, phosphate-buffered saline (PBS), or 0.67mM human serum albumin (HSA) solutions. , 0.25, 0.5, and 1mM), respectively (see Table 1).

[Table 1]

*Values are expressed as mean SD (n = 3)

Referring to Table 1, among the three synthesized compounds, Gd-galangin showed the highest r ₁ relaxivity, while Gd-flavone, Gd-BT-DO3A, and Gd-DOTA showed similar values. In addition, in a solution containing 0.67mM HSA, it was confirmed that Gd-chrysin showed a slightly higher level of r ₁ relaxivity than other compounds.

Meanwhile, to calculate the relative polarity of Gd-flavone, Gd-chrysin, and Gd-galangin, the octanol-water partition coefficient (log P) was calculated using a known method, and the values were -1.40, -0.91, and -0.74, respectively. was obtained (see Table 1). For comparison, the log P values of clinically used drugs Gd-BT-DO3A (-3.13) and Gd-DOTA (-3.09) were also calculated.

As a result, it was confirmed that the lipophilicity of the new gadolinium complex of the present invention was significantly higher than that of the gadolinium complex currently used in clinical settings. This is due to the presence of polyphenol hydroxy groups present in the compounds of the present invention. Considering that log P values are highly correlated with relaxivity and protein binding, it can be seen that the high relaxivity of Gd-flavone, Gd-chrysin, and Gd-galangin is related to high lipophilicity.

Dynamic stability evaluation

Endogenous metal ions, including Zn ²⁺ , Cu ²⁺ and Ca ²⁺ , can compete with Gd ³⁺ ions for chelation, resulting in Gd ion loss, leading to diseases such as nephrogenic systemic fibrosis (NSF) or in the brain. Diseases such as Gd ³⁺ ion deposition can cause it.

Compared to linear chelates such as Gd-DTPA and Gd-DTPA-EOB, Gd-based macrocyclic chelates are characterized by higher kinetic inactivity. Referring to Figure 5(a), among the compounds evaluated, Gd-galangin showed the highest kinetic inertness value, and Gd-galangin, which has the same macrocyclic chelate structure as Gd-BT-DO3A and Gd-DOTA, flavone and Gd-chrysin showed similar values. Additionally, when the R ₂ relaxation rate was monitored for 72 hours, the linear DTPA analog showed a significant decrease in R ₂ values.

In addition, the R ₂ relaxivity-related stability of Gd-flavone, Gd-chrysin, and Gd-galangin over time was evaluated at various pH values from pH 1 to 11 and Gd-BT-DO3A as a control. 5(b)-(d) showing the results, it can be seen that the R ₂ values of Gd-flavone, Gd-chrysin, and Gd-galangin remain relatively constant over a wide range of pH values from pH 3 to 11. It was confirmed that the synthesized gadolinium complex was sufficiently stable under both acidic and basic conditions. These results confirm that Gd-flavone, Gd-chrysin, and Gd-galangin maintain sufficient stability in vivo.

Experimental Example 2: Solubility evaluation of gadolinium-flavonoid complex

According to the example, the insoluble substances 7-hydroxyflavone, chrysin, and galangin were synthesized into a water-soluble gadolinium complex, and the solubility for use as an intravenous injection preparation was evaluated.

[Table 2]

Table 2 above shows the results of evaluating the water solubility of the flavonoids 7-hydroxyflavone, chrysin, and galangin and the materials synthesized with water-soluble gadolinium complexes of 7-hydroxyflavone, chrysin, and galangin at 25°C.

As shown in Table 2, flavonoids 7-hydroxyflavone, chrysin, and galangin are insoluble substances that are almost insoluble in water. Because these substances are difficult to absorb in the body and have low bioavailability, it is difficult to expect anti-inflammatory, antioxidant, and anti-cancer effects from these substances.

On the other hand, in the case of water-soluble gadolinium complexes, everything can be dissolved beyond 50mM, which is mainly used to conduct contrast agent-related experiments. In particular, Gd-galangin was shown to be dissolved and maintained up to 100mM.

Figure 6 shows images of the synthesized water-soluble gadolinium complexes observed immediately after dissolution in tertiary distilled water, saline solution, and PBS, 30 minutes after dissolution, and 2 hours after dissolution. From the left, the order is Gd-galangin, Gd-chrysin, and Gd-flavone.

Looking at Figure 6, all three types of synthesized gadolinium complexes were dissolved immediately after dissolution, but after 30 minutes, Gd-flavone precipitated cloudily. After 2 hours after dissolution, Gd-chrysin dissolved in distilled water and PBS began to precipitate, and the precipitation rate was relatively slow in saline solution. Gd-galangin existed as a solution without precipitating even after 7 days, and showed excellent solubility compared to Gd-chrysin and Gd-flavone.

Maintaining the aqueous solution state is also related to the stability of the material. The three types of synthesized gadolinium complexes had similar structures, but only Gd-galangin maintained high solubility. Through these results, it was confirmed that Gd-galangin is most suitable as an injection preparation.

Gd-galangin is an insoluble substance, and can be administered intravenously by increasing solubility by synthesizing it into a gadolinium complex. When used as an intravenous preparation, direct drug delivery is possible, and anti-inflammatory, antioxidant, and anticancer effects can be expected to increase.

Experimental Example 3: Cytotoxicity test of gadolinium-flavonoid complex

In a toxicity test on RAW 264.7 cells, three types of flavonoids (7-hydroxyflavone, chrysin, galangin) and three types of gadolinium complexes synthesized as moieties (Gd-flavone, Gd-chrysin, Gd-galangin) were compared.

Looking at Figure 7 showing the results, the toxicity results for drug concentrations from 0 to 100 uM Gd-flavone and Gd-chrysin synthesized according to an embodiment of the present invention show similar or even increased results to 7-hydroxyflavone and chrysin. It shows. However, Gd-galangin shows differences from galangin without showing toxicity at 50 uM.

Likewise, in the cytotoxicity test, Gd-galangin showed the lowest level among the three flavonoids and three gadolinium complexes. These results show that among the synthesized gadolinium complexes, Gd-galangin is the most suitable for clinical use.

Experimental Example 4: Evaluation of antioxidant efficacy of gadolinium-flavonoid complex

DPPH, FRAP, ABTS free radical scavenging activity in vitro experiments

Free radicals are harmful substances that destroy cells in the body, and their relationship with inflammation is well known. Antioxidant action that removes large amounts of free radicals is very important. DPPH, FRAP, and ABTS experiments were conducted to evaluate free radical scavenging activity. This is an experiment in which a highly reactive reagent with a single electron generates free radicals and compares them by measuring the difference in wavelength absorption using a spectrophotometer when reacting with a drug. Representative antioxidants ascorbic acid (AA) and trolox (TR) were used as controls.

Referring to Figure 8, while neither Gd-flavone nor Gd-chrysin can scavenge radicals, Gd-galangin has a radical scavenging activity similar to that of ascorbic acid (AA) and Trolox (TR) in FRAP and ABTS. showed. This result shows that Gd-galangin has the most excellent antioxidant effect.

Comparison of free radical removal effects in vivo experiments

The luminol derivative L-012 is a highly sensitive chemiluminescent material characterized by greater activity than luminol itself. Although it reacts with various ROS generated in the body, its activation time is limited. Therefore, L-012 was administered repeatedly to compare the efficacy of the drug, and the free radical removal effect was compared.

Looking at Figure 9 showing the results, the ROS scavenging ability (ROS Scavenging Affinity) generated by LPS was significantly reduced in Gd-galangin compared to saline, demonstrating ROS Scavenging Affinity in vivo. did.

Experimental Example 5: Evaluation of anti-inflammatory efficacy of Gd-galangin

In vitro experiments on LPS-induced iNOS and NO inhibition

iNOS (inducible nitric oxide synthase) is a protein induced by inflammatory cytokines expressed in various cells during inflammatory reactions. It performs oxidative reduction and produces NO (nitric oxide). NO performs various physiological functions such as vasodilation, inhibition of platelet aggregation, and neurotransmission, and also plays an important role in inflammatory reactions.

The effect of LPS on NO production in Raw cells by iNOS expressed in macrophages and Kupffer cells was confirmed.

As a result, as shown in Figure 10, Gd-galangin decreased the NO increased by LPS from 50 uM, and iNOS tended to decrease depending on the concentration. This result shows that Gd-galangin decreases the NO increased by LPS by reducing iNOS expression. It shows that it contributes to the inhibition of induced NO production.

In vitro experiments on inhibition of NLRP3 inflammasome activity

Meanwhile, in order to evaluate the inhibition ability of Gd-galangin in relation to NLRP3 inflammasome activation, its ability to downregulate the expression of ASC, IL-1β, and NLRP3 was confirmed, and the results were analyzed. It is shown in Figure 11.

The results show that treatment of RAW cells with LPS alone increased the expression of NLRP3 and ASC, which can promote NLRP3 inflammasome activation. Administration of Gd-galangin appears to slightly reduce LPS-induced NLRP3 expression, while ASC and IL-1β produced in response to LPS treatment decreased in a concentration-dependent manner. These results show that Gd-galangin has an anti-inflammatory effect by suppressing the expression of NLRP3 and ASC and their pro-inflammatory product, IL-1β.

In vivo experiments on NLRP3, ASC, and iNOS

Figure 12 shows the results of Western blot on inflammatory tissue in an LPS-induced myositis animal model. Similar to the results of in vitro experiments, LPS promoted the expression of iNOS and NLRP3 inflammasome-related factors, and administration of Gd-galangin promoted the expression of these factors. Anti-inflammatory efficacy was also demonstrated in vivo by confirming that the inflammatory response was suppressed.

Comparison of anti-inflammatory effects of galangin and Gd-galangin in vivo experiment

H&E

Figure 13 shows the results of histological evaluation by H&E staining on inflammatory tissue induced by LPS. Looking at Figure 13, it can be seen that the intercellular spacing is very tight in the case of normal cells compared to the LPS-administered group. On the other hand, compared to the LPS only group, both the intravenous injection of Gd-galangin and the oral administration of galangin showed a better condition, but the intercellular spacing was wider in galangin overall, so the group in which Gd-galangin was administered intravenously This showed results closer to the normal tissue state.

Immunohistochemistry (IHC)

Figure 14 shows the results of an in vivo experiment to evaluate the anti-inflammatory efficacy of galangin and Gd-galangin, showing the results of immunohistochemical staining. If the target protein to be observed by immunohistochemical staining (IHC), a specific detection method using an antigen-antibody reaction, is an antigen, the antibody against the target protein specifically recognizes and binds only to that protein. To observe this, a marker is added and color is developed to detect it.

Looking at Figure 14, it can be seen that ASC, iNOS, NLRP3, and IL-1β are all expressed when inflammation is induced by LPS. Afterwards, it can be seen that the Gd-galangin group has a clear decrease compared to the galangin group. This shows that intravenous injection of Gd-galangin with increased solubility can produce anti-inflammatory and antioxidant effects more quickly and effectively than orally administered galangin.

Experimental Example 6: In vivo evaluation of MR diagnostic target in inflammation of Gd-galangin

The diagnostic imaging properties of Gd-galangin were evaluated using a mouse model of LPS-induced inflammation. 24 hours after LPS injection, Gd-galangin and Gd-BT-DO3A (control group) were injected intravenously at the same concentration (0.1 mmol Gd/kg) and coronal and axial injections were performed throughout the body and at the inflamed area at 3.0 T. ) T1-weighted MR images were obtained.

15(a) and (b), MR image contrast of liver and inflammatory lesions showed significant differences between Gd-galangin and Gd-BT-DO3A treated mice. Gd-galangin maintained high signal intensity for up to 3 hours. This pattern of enhancement is consistent with the biodistribution results (Figure 15(d)).

The signal intensity of the gallbladder was enhanced 1 hour after intravenous injection, and during the hepatobiliary phase, the contrast agent was absorbed by hepatocytes and then excreted through the biliary route, showing a characteristic enhancement pattern for hepatobiliary excretion contrast agent.

The flavonoid moiety of the gadolinium complex confers high lipophilicity, and Gd-galangin can maintain higher and longer signal intensity in the liver and gallbladder.

The signal enhancement of Gd-galangin measured based on CNR at the site of inflammation continued to increase for up to 1.5 h, resulting in a CNR difference approximately 5 times stronger than that obtained using Gd-BT-DO3A. Additionally, signal enhancement in inflamed tissue was maintained for at least 3 hours (Figure 15(c)).

Due to the high lipophilicity of Gd-galangin, its residence time in the body was extended and the targeting ability of this agent to inflamed tissue was improved, confirming favorable results for inflammation diagnosis using MRI.

Experimental Example 7: Evaluation of anti-inflammatory efficacy of Gd-galangin for hepatitis

Hepatitis is an inflammation of the liver cell tissue and includes acute hepatitis caused by viruses and toxins, and chronic hepatitis such as alcoholic and non-alcoholic hepatitis. There are many treatment methods, but treatment for inflammation is the most important. Targeted treatment for inflammation is very important because it can slow or prevent progression to chronic disease through inflammation treatment.

Metabolic disorders are associated with increased oxidative stress and DNA damage, which induces PARP activity. Overactivation of PARP can cause ATP and nicotinamide adenine dinucleotide (NAD+) depletion, leading to cell death. PARP expression is associated with several hepatitis diseases and is used as an indicator to detect cell death.

Referring to Figure 16, with respect to intravenous injection of Gd-galangin and oral administration of galangin to an animal model of acute hepatitis induced by LPS and D-galactosamine, there was no effect on the inflammatory factors NLRP3 and PARP, nor on galangin. It showed no effect and showed statistically valid results for Gd-galangin (0.1 mmol/kg).

Effect of Gd-galangin on LPS-induced MAPK signaling pathway

The effects of Gd-galangin on the MAPK signaling pathway were investigated. Phosphorylation of Erk, JNK, and p38 was detected in response to LPS stimulation of RAW 264.7 cells, which was generally maintained at high levels for 30 minutes to 1 hour (see Figure 17). However, at 3 h after treatment, all three of these MAPK pathway members showed low levels of phosphorylation.

As shown in Figure 17(a)-(c), administration of Gd-galangin did not significantly change the levels of phosphorylated Erk and p38, but effectively promoted a decrease in the level of phosphorylated JNK. These results imply that the anti-inflammatory activity of Gd-galangin is mediated, at least in part, through inhibition of MAPK signaling, especially in relation to JNK.

Anti-inflammatory effect of Gd-galangin through inhibition of NF-kB translocation and IkBα phosphorylation

18(a)-(c), in response to LPS-treated RAW 264.7 cells, a lower level of NF-kB and a tendency to accumulate in the cytoplasm were confirmed compared to untreated cells. This indicates that LPS stimulation promotes nuclear translocation of NF-kB. On the other hand, in response to Gd-galangin treatment, we found that cytoplasmic levels of NF-kB were maintained, whereas there was a significant decrease in the nucleus. This indicates that Gd-galangin inhibits nuclear translocation of NF-kB.

Meanwhile, looking at Figure 18(c), while LPS promoted phosphorylation of IkBα, low levels of phosphorylated IkBα were detected after administration of Gd-galangin. These results indicate that Gd-galangin inhibits nuclear translocation of NF-kB through its inhibitory effect on the phosphorylation of IkBα.

Anti-inflammatory effects of Gd-galangin through Nrf2 and HO-1 expression

In response to oxidative stress, Nrf2 mediates HO-1 upregulation to activate antioxidant responses. Accordingly, the relevance of Gd-galangin to the Nrf2 signaling pathway was investigated, and the results are shown in Figure 19.

Referring to Figure 19(a), the elevated phosphorylation of Nrf2 stimulated by LPS was further increased by Gd-galangin treatment. After 12 hours of LPS stimulation, a clear and statistically significant difference was detected between the LPS-only treated group and the Gd-galangin-treated group.

Meanwhile, with regard to HO-1 expression, there was no significant difference between the LPS-only treatment group and the Gd-galangin treatment group up to 6 hours after LPS stimulation, but HO-1 expression and HO-1 expression after 12 hours in response to phosphorylation of Nrf2. Relatedly, a more significant increase was observed in Gd-galangin treated cells (Figure 19(b)).

These results indicate that Gd-galangin has an antioxidant effect by promoting phosphorylation of Nrf2 and upregulated expression of HO-1.

conclusion

The present invention evaluated the diagnostic ability of a gadolinium-based contrast agent combined with flavonoids for inflammation. Flavonoids and their derivatives are known to be effective in preventing and treating inflammatory diseases such as encephalitis, hepatitis, rheumatism, and serious diseases such as cancer, Alzheimer's disease, and Parkinson's disease. However, because most flavonoids have very low solubility or are insoluble in water, conventional flavonoid substances have been administered by oral administration. Oral administration is the most preferred method of drug administration due to convenience, patient compliance, and reduced risk of cross-infection.

Oral drugs are mostly absorbed through digestion or metabolism, and their absorption into the body is very limited. As such, there is a problem in that it exhibits limited bioavailability due to low permeability and stability, seriously reducing its effectiveness as a therapeutic agent.

To solve this problem, the present invention combines a DO3A chelating substance and a flavonoid to increase water solubility, and the high lipophilicity due to the binding portion can increase intracellular diffusion ability.

Due to the improved solubility and lipophilicity of the synthesized DO3A-galangin, intravenous injection is possible, and the rapid and immediate anti-inflammatory effect of the flavonoid can be achieved even with a small amount of injection.

Looking at Figure 20, among the basic skeleton of flavonoids, the 4-carbonyl group, C2=C3 double bond, and hydroxyl group at the C3 position play a very important role in radical removal and antioxidant function through synergistic effect. Ring B is due to conjugation with the chromone moiety, which provides more resonance and binding sites and supports the chromone.

The linking group synthesis utilized the unique high reactivity of the hydroxyl group at position 7, and the gadolinium compound of the present invention was synthesized in high yield by forming an ester bond with DO3A through the linking group.

Referring to Figure 1, DO3A-flavonoid was designed to maintain active sites for antioxidant effects and increase synthesis efficiency. The ester bond-based DO3A and flavonoid binding portion has a large structure that is difficult for esterases to act on, so it is protected when decomposed, and as a result, the synthesized material has high stability.

Meanwhile, flavone, chrysin, and galangin differ in the position and number of hydroxyl groups for rings A and C. Considering the structural features mentioned above, the greatest effect can be expected from galangin, which was confirmed in radical scavenging ability tests (DPPH, FRAP, ABTS) conducted with the newly synthesized structure. In particular, the results for Gd-flavone and Gd-chrysin show the importance of the hydroxyl group at position 3.

Based on this, the antioxidant and anti-inflammatory effects of Gd-galangin on LPS-induced inflammation were confirmed in vitro and in vivo.

The proposed mechanism underlying the effects of Gd-galangin is summarized in Figure 21. The inflammatory response is an important response for survival dominated by inflammatory cytokines and chemokines. Gd-galangin of the present invention suppresses iNOS and NLRP3 inflammasome expression and regulates the expression of downstream factors NO and IL-1β. In particular, NLRP3 inflammasome activation is a target for inflammation treatment because it promotes inflammation and induces disease development. IL-1β is an inflammatory cytokine that is matured by activated NLRP3 inflammasome. Gd-galangin reduces IL-1β expression due to its contribution to NLRP3 and ASC suppression.

Under normal conditions, I _k B binds to NF- _k B and inhibits its translocation to the nucleus. In contrast, under LPS-induced stress conditions, phosphorylation of _IkB triggers the release of NF- _kB , restoring its nuclear translocation. Gd-galangin also shows that inhibition of phosphorylation of _IkB inhibits NF- _kB translocation to the nucleus. Additionally, in response to oxidative stress, Nrf2 mediates the upregulation of HO-1 to initiate antioxidant activity. This upregulated expression of HO-1 contributes to the maintenance of redox homeostasis through several mechanisms. In this process, Keap1 generally binds to Nrf2 and inhibits its translocation to the nucleus. Phosphorylation of Nrf2 promotes its dissociation from Keap1, which contributes to the expression of HO-1. Gd-galangin exerts antioxidant effects by promoting phosphorylation of Nrf2 and upregulating HO-1 expression.

Meanwhile, the MAPK signaling pathway is a cascade of serine/threonine kinases that regulate cell survival and death, and Erk, JNK, and p38 are members of the MAPK family that contribute to LPS-stimulated inflammation by phosphorylation. Gd-galangin tended to inhibit Erk, JNK, and p38, and showed particularly high anti-inflammatory activity against JNK.

Through the above experiments, the antioxidant and anti-inflammatory effects of Gd-galangin synthesized according to the present invention were sufficiently proven, and considering that inflammation is an important factor in various diseases, Gd-galangin has therapeutic efficacy for various inflammatory diseases. can indicate.

In particular, Gd-galangin, used as a T ₁ MR contrast agent according to the present invention, showed a contrast enhancement effect up to 5 times better than conventional contrast agents for hepatitis, an inflamed tissue, and maintained its durability for more than 3 hours. These characteristics resulted from the combination of galangin and gadolinium chelate, which supported its diagnostic ability for inflammation.

Although the present invention has been described above with reference to preferred embodiments, those skilled in the art can make various modifications and changes to the present invention without departing from the spirit and scope of the present invention as set forth in the following patent claims. You will understand that it is possible.

Claims (11)

Compound represented by the following formula (1):

(One) here, L is *-(CH ₂ ) _x -A ¹ -(CH ₂ ) _y -A ² -(CH ₂ ) _z -*, x, y and z are each independently selected as random integers from 0 to 5, A ¹ and A ² are single bonds, in the group containing *-COO-*, *-CO-*, *-NH-*, *-CH ₂ -*, *-CONH-* and *-O-* Each is one or more independently selected structures, X is a structure having the following formula (2):

(2) Here, R ₁ and R ₂ represent a hydroxy group, and * is a binding site. According to paragraph 1, Compounds represented by the following formula (3):

(3) Here, R ₁ and R ₂ represent a hydroxy group. According to paragraph 1, The gadolinium (Gd) coordinates with one or more water molecules, compound. An MRI contrast agent comprising the compound according to any one of claims 1 to 3. According to paragraph 4, The MRI contrast agent is characterized in that it targets diagnosis and treatment of hepatitis. MRI contrast agent. According to clause 4, The MRI contrast agent is administered intravenously, MRI contrast agent. According to paragraph 4, The MRI contrast agent has anticancer activity and is characterized in that it targets cancer diagnosis and treatment, MRI contrast agent. An anti-inflammatory agent for intravenous administration comprising the compound according to any one of claims 1 to 3, which targets an inflammatory site and has anti-inflammatory activity at the inflammatory site. According to clause 8, The anti-inflammatory agent is characterized in that it targets diagnosis and treatment of the inflamed area, Anti-inflammatory medication for intravenous administration. According to clause 8, The anti-inflammatory agent specifically targets hepatitis and exhibits anti-inflammatory activity against hepatitis. Anti-inflammatory medication for intravenous administration. An antioxidant for intravenous administration comprising the compound according to any one of claims 1 to 3 and having antioxidant efficacy.

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